Mu opioid receptor agonist analogs of the endomorphins

ABSTRACT

The invention relates to cyclic peptide agonists that bind to the mu (morphine) opioid receptor and their use in the treatment of acute and/or chronic pain. Embodiments of the invention are directed to cyclic pentapeptide and hexapeptide analogs of endomorphin that have (i) a carboxy-terminal extension with an amidated amino acid and (ii) a D-amino acid substitution in position 2. These peptide analogs exhibit decreased tolerance relative to morphine, increased solubility compared to similar tetrapeptide analogs, while maintaining favorable or improved therapeutic ratios of analgesia to side effects.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.14/268,057, filed on May 2, 2014, which is a continuation of U.S.application Ser. No. 13/477,423, filed on May 22, 2012, now U.S. Pat.No. 8,716,436, which is a continuation-in-part of PCT/US2011/43306,filed on Jul. 8, 2011, which claims the benefit of U.S. ProvisionalApplication Ser. No. 61/363,039, filed on Jul. 9, 2010, each of which isincorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

A portion of the work described herein was supported by a Senior CareerResearch and Merit Award, Grant No. I01BX001489 from the Department ofVeteran Affairs, Grant No. DM090595 from the Department of Defense, andGrant No. N00014-09-is 1-0648 from the Office of Naval Research of theDepartment of Defense. The United States government has certain rightsin this invention.

INCORPORATION OF SEQUENCE LISTING

The biological sequence information in this application is included inan ASCII text file having the file name “TU386CIPSEQ.txt”, created onAug. 24, 2012, and having a file size of 3,011 bytes, which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to peptide agonists that bind to the mu(morphine) opioid receptor and their use in the treatment of acute andchronic pain.

BACKGROUND OF THE INVENTION

Activation of the mu opioid receptor is among the most effective meansof alleviating a wide range of pain conditions. Of the recently clonedopioid receptors e.g., mu opioid receptor (“MOR”, Ref. 3,20,21), deltaopioid receptor (“DOR”, Ref 6,9), and kappa (“KOR”, Ref 12-14), the vastmajority of clinically used opioids act at the mu receptor. Asillustrated in genetically altered “knock-out” mice, the absence of themu receptor eliminates the analgesic effects of morphine (8),illustrating its central role in opioid-induced pain relief. The uniqueeffectiveness of mu agonists can be attributed to several factors,including their presence in numerous regions of the nervous system thatregulate pain processing and activation of multiple mechanisms thatlimit pain transmission (e.g., inhibiting release of excitatorytransmitters from the peripheral nervous system and decreasing cellularexcitability in the central nervous system).

Limitations on the use of opioids result from negative side effects,including abuse liability, respiratory depression, and cognitive andmotor impairment. Major efforts to develop compounds that maintainanalgesic properties while reducing the negative side effects have metwith limited success. This is evident from the recent epidemic ofprescription drug abuse. Numerous attempts at targeting alternativemechanisms of pain relief to avoid these side effects have generallybeen met with similar problems, i.e., a profile of adverse effects thatare different from opioids, but often sufficiently serious to warrantremoval from the market (e.g., COX inhibitors) or lack of approval toenter the market (e.g., TRP receptor antagonists). Over 100 millionpatients annually in the United States experience acute or chronic painand frequently do not achieve adequate relief from existing drugs due tolimited efficacy or excessive side effects. Elderly patients tend toshow greater sensitivity to severe pain and recent guidelines of theAmerican Geriatric Society suggest greater use of opioids and reductionof non-steroidal anti-inflammatory drugs (NSAIDs) (10). Impairment ofmotor and cognitive function can be more debilitating in the elderlythan in younger patients, particularly due to increased risk offractures (7). Opioids with reduced motor and cognitive impairment aretherefore a growing unmet need.

Natural endogenous peptides from bovine and human brain that are highlyselective for the mu opioid receptor relative to the delta or kappareceptor have been described (23 and U.S. Pat. No. 6,303,578 which isincorporated herein by reference in its entirety). These peptides arepotent analgesics and have shown promise of reduced abuse liability (22)and respiratory depression (4,5), as measured in rodent studies. Thelimited metabolic stability of the natural peptides led to thedevelopment of cyclized, D-amino acid-containing tetrapeptide analogs ofthe endomorphins (U.S. Pat. No. 5,885,958 which is incorporated hereinby reference in its entirety) of sufficient metabolic stability toproduce potent analgesia in rodents after peripheral administration. Alead compound from this group reportedly was 3-fold more potent thanmorphine in alleviating neuropathic pain and showed reduced rewardingproperties in animal models that are correlated with abuse potential.While these results are promising, the development of additionalcompounds showing equal or better properties is desirable. The instantinvention addresses this need by providing peptide analogs havingunexpectedly better solubility and side-effect profiles than thepreviously described materials.

SUMMARY OF THE INVENTION

Metabolically stable analogs of the endomorphins, endogenous opioidshighly selective for the Mu opioid receptor (MOR) are described herein.Compared to morphine, the pentapeptide and hexapeptide compounds (EManalogs) of Formula I showed dramatically improvedanalgesia-to-side-effect ratios. At doses providing equal or greaterantinociception compared to morphine in the rat, the analogs showedreduced (a) respiratory depression, (b) impairment of motorcoordination, (c) tolerance and hyperalgesia, (d) glial p38/CGRP/P2X7receptor signaling, and (e) reward/abuse potential in both conditionedplace preference and self-administration tests. Differential effects onglial activation indicate a mechanism for the relative lack of sideeffects by the analogs compared to morphine. The results indicate thatEM analogs of Formula I provide excellent pain relief mediated byselective MOR activation, but with remarkably safer side effect profilescompared to opioids like morphine.

The EM analogs compounds of Formula I, described below, provideantinociceptive effects equal or greater than morphine with lessrespiratory depression, motor impairment, tolerance, immune reactivity,and reward/abuse liability, relative to morphine. Opioids, and morphinein particular, induce proinflammatory glial cell, CGRP, or P2X7 receptoractivation, which leads to undesirable tolerance to chronic opioidadministration, and the need to increase dosages for effective painrelief. The analogs of Formula I do not induce proinflammatory glialcell, CGRP, or P2X7 receptor activation, and thus avoid the tolerance tochronic administration that is associated with opioids. Overall, the EManalogs of Formula I represent a major advance for pain research bysurprisingly providing equally effective analgesia compared to morphine,with absent or substantially reduced side effects.

An embodiment of the instant invention is directed to pentapeptide andhexapeptide analogs of endomorphins that differ from the previouslydescribed tetrapeptide analogs by having (i) a carboxy-terminalextension with an amidated amino acid, (ii) side-chain to side-chaincyclization, and (iii) in some embodiments, a substitution in position2. The pentapeptide and hexapeptide analogs of the present inventionexhibit increased solubility relative to the tetrapeptides whilemaintaining favorable therapeutic ratios of analgesia-to-side effects.

The compounds of the present invention are cyclic peptides that act asmu opioid receptor agonists with high affinity. These compounds providerelief of acute pain, chronic pain, or both, and comprise or consist ofcompounds of Formula I:

(I) H-Tyr-cyclo[X₁-X₂-X₃-X₄]-X₅. X₁ and X₄ each independently is anacidic amino acid (i.e., an amino acid comprising a carboxylicacid-substituted side-chain) or a basic amino acid (i.e., an amino acidcomprising an amino-substituted side-chain), with the proviso that if X₁is an acidic amino acid (e.g., D-Asp or D-Glu), then X₄ is a basic aminoacid (e.g., Lys, Orn, Dpr, or Dab), and vice versa. Preferably, X₁ isD-Asp, D-Glu, D-Lys, D-Orn, D-Dpr or D-Dab; while X₄ preferably is Asp,Glu, Lys, Orn, Dpr or Dab. X₂ and X₃ each independently is an aromaticamino acid (i.e., an amino acid comprising an aromatic group in the sidechain thereof). For example, X₂ preferably is Trp, Phe, or N-alkyl-Phe,where the alkyl group preferably comprises 1 to about 6 carbon atoms,i.e., a (C₁ to C₆) alkyl group. X₃ preferably is Phe, D-Phe, or p-Y-Phewhere Y is NO₂, F, Cl, or Br. X₅ is selected from the group consistingof —NHR, Ala.-NHR, Arg-NHR, Asn-NHR, Asp-NHR, Cys-NHR, Glu-NHR, Gln-NHR,Gly-NHR, His-NHR, Ile-NHR, Leu-NHR, Met-NHR, Orn-NHR, Phe-NHR, Pro-NHR,Ser-NHR, Thr-NHR, Trp-NHR, Tyr-NHR, and Val-NHR; where R is H or analkyl group (e.g. a (C₁ to C₁₀) alkyl group such as methyl, ethyl,propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl, isohexyl,heptyl, or isoheptyl). The peptide of Formula I is cyclic (shown as“c[X₁-X₂-X₃-X₄]” or “cyclo[X₁-X₂-X₃-X₄]” in the formulas describedherein) by virtue of an amide linkage between the carboxylic acid andamino substituents of the side chains of amino acid residues X₁ and X₄.For example, the linkage can be an amide bond formed between the sidechain amino group of the D-Lys, D-Orn, D-Dpr, D-Dab, Lys, Orn, Dpr, orDab with the side chain carboxyl group of D-Asp, D-Glu, Asp, or Glu.

In one embodiment of the invention directed to a peptide of Formula I,X₅ is NHR, R is H, and X₅ can be —NH₂ (i.e., the peptide is an amidatedpentapeptide), or Ala-NH₂, Arg-NH₂, Asn-NH₂, Asp-NH₂, Cys-NH₂, Glu-NH₂,Gln-NH₂, Gly-NH₂, His-NH₂, Ile-NH₂, Leu-NH₂, Met-NH₂, Orn-NH₂, Phe-NH₂,Pro-NH₂, Ser-NH₂, Thr-NH₂, Trp-NH₂, Tyr-NH₂, or Val-NH₂, (i.e., thepeptide is an amidated hexapeptide). In one particular embodiment, X₅ isNH₂. In other particular embodiments, X₅ is Ala-NH₂, Arg-NH₂, Asn-NH₂,Asp-NH₂, Cys-NH₂, Glu-NH₂, Gln-NH₂, Gly-NH₂, His-NH₂, Ile-NH₂, Leu-NH₂,Met-NH₂, Orn-NH₂, Phe-NH₂, Pro-NH₂, Ser-NH₂, Thr-NH₂, Trp-NH₂, Tyr-NH₂,or Val-NH₂.

Another embodiment of the invention is directed to a peptide of FormulaI, wherein X₁ is D-Asp, D-Glu, D-Lys, or D-Orn; and X₄ is Asp, Glu, Lys,or Orn.

Another embodiment of the invention is directed to a compound of FormulaI, wherein X₅ is NHR and R is a (C₁ to C₁₀) alkyl.

Another embodiment of the invention is directed to a peptide of FormulaI, wherein the aromatic amino acid of X₂ is Trp, Phe, or N-alkyl-Phe,and the alkyl group of N-alkyl-Phe is a (C₁ to C₆) alkyl. In oneparticular embodiment, X₂ is N-methyl-Phe (N-Me-Phe).

Another embodiment of the invention is directed to a peptide of FormulaI, wherein the aromatic amino acid residue of either X₂ or X₃ is Phe,D-Phe, Trp, D-Trp, D-Tyr, N-alkyl-Phe, and the alkyl group ofN-alkyl-Phe is (C₁ to C₁₀) alkyl or p-Y-Phe, wherein Y is NO₂, F, Cl, orBr.

Another embodiment of the invention is directed to a peptide of FormulaI, wherein the aromatic amino acid of X₃ is Phe, D-Phe, or p-Y-Phe,wherein Y is NO₂, F, Cl, or Br. In one particular embodiment, X₃ isp-Cl-Phe.

Another embodiment of the invention is directed to a peptide of FormulaI selected from the group consisting of Tyr-c[D-Lys-Trp-Phe-Glu]-NH₂(SEQ ID NO:1); Tyr-c[D-Glu-Phe-Phe-Lys]-NH₂ (SEQ ID NO:2);Tyr-c[D-Lys-Trp-Phe-Glu]-Gly-NH₂ (SEQ ID NO:3);Tyr-c[D-Glu-Phe-Phe-Lys]-Gly-NH₂ (SEQ ID NO:4);Tyr-c[D-Lys-Trp-Phe-Asp]-NH₂ (SEQ ID NO:5);Tyr-c[D-Glu-N-Me-Phe-Phe-Lys]-NH₂ (SEQ ID NO:6); andTyr-c[D-Orn-Phe-p-Cl-Phe-Asp]-Val-NH₂ (SEQ ID NO:7).

Another aspect of the invention is directed to a pharmaceuticalcomposition comprising a peptide of Formula I and a pharmaceuticallyacceptable carrier (e.g., a diluent or excipient).

Yet another aspect of the invention is directed to the use of a peptideof Formula I in a method of treating a patient having a condition thatresponds to an opioid, or a condition for which opioid treatment isstandard in the art. Such a method comprises or consists ofadministering to the patient an effective amount of a peptide of FormulaI of the invention. Particular embodiments of this method can befollowed for the purpose of providing at least one effect selected from(i) analgesia (pain relief), (ii) relief from a gastrointestinaldisorder such as diarrhea, (iii) therapy for an opioid drug dependence,and (iv) treatment of any condition for which an opioid is indicated. Insome embodiments the peptides of Formula I can be used to treat acute orchronic pain. Uses for the peptides of Formula I also include, but arenot be limited to, use as antimigraine agents, immunomodulatory agents,immunosuppressive agents or antiarthritic agents. Certain embodiments ofthe methods of the present invention, such as treatment of pain oropioid drug dependence, are directed to patients having a history ofopioid substance abuse. In certain embodiments of the present methods,the peptide is administered parenterally (e.g., intravenous). Thisinvention also relates to a peptide of Formula I for use in one of saidmethods of treatment.

Another aspect of the invention is directed to a method of activating orregulating a mu-opioid receptor by contacting the mu-opioid receptorwith a compound of the invention, as well as the use of the peptide ofFormula I in such a treatment.

Another aspect of the invention is directed to a method of measuring thequantity of a mu opioid receptor in a sample using a peptide of FormulaI. This method can comprise or consist of the following steps: (i)contacting a sample suspected of containing a mu opioid receptor with apeptide of Formula I to form a compound-receptor complex, (ii) detectingthe complex, and (iii) quantifying the amount of complex formed.

Another aspect of the invention is directed to the use of a peptide ofFormula I to perform a competitive assay method of detecting thepresence of a molecule that binds to a mu opioid receptor. This methodcan comprise or consist of the following steps: (i) contacting a samplesuspected of containing a molecule that binds to a mu opioid receptorwith a mu opioid receptor and a peptide of Formula I, wherein thecompound and receptor form a compound-receptor complex; (ii) measuringthe amount of the complex formed in step (i); and (iii) comparing theamount of complex measured in step (ii) with the amount of a complexformed between the mu opioid receptor and the peptide in the absence ofsaid sample.

Six critical side effects of currently used opioids were surprisinglyreduced or absent with the compounds of Formula I: abuse liability,respiratory depression, motor impairment, tolerance, hyperalgesia, andglial activation. This extensive profile of low side effects forpeptides shown to cross the blood brain barrier and provide prolongedantinociception is unprecedented. The lack of respiratory depression andmotor impairment provides an improved safety profile. The lack of glialactivation by a mu agonist, where morphine induces a proinflammatoryresponse, is a novel finding that suggests both a mechanism for thereduced side effects of the analogs and a therapeutic profile withreduced inflammatory complications. The associated reduction intolerance indicates improved long-term effects. The absence of CPP, andespecially SA in the sensitive long-access paradigm, indicates abuse isunlikely. Pain therapy with opioids continues to present troublingquestions for doctors and patients who must weigh the risk of causingadverse side effects with effectively alleviating pain. This strugglecould be significantly reduced by development of novel opioids with thepotent analgesia and low side-effect profile shown by the EM analogcompounds of Formula I.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Tyr-c[D-Lys-Trp-Phe-Glu]-NH₂ (SEQ ID NO:1), which isdescribed as “Compound 1” in the following disclosure. The structuraland basic molecular formulae, as well as the molecular weight (MW), areshown for Compound 1.

FIG. 2 shows Tyr-c[D-Glu-Phe-Phe-Lys]-NH₂ (SEQ ID NO:2), which isdescribed as “Compound 2” in the following disclosure. The structuraland basic molecular formulae, as well as the molecular weight (MW), areshown for Compound 2.

FIG. 3 shows Tyr-c[D-Glu-Phe-Phe-Lys]-Gly-NH₂ (SEQ ID NO:4), which isdescribed as “Compound 4” in the following disclosure. The structuraland basic molecular formulae, as well as the molecular weight (MW), areshown for Compound 4.

FIG. 4 shows opioid receptor binding activity for Compound 1. (A) mureceptor binding of “Compound 1” (triangles) or DAMGO (squares). (B)Antagonist activity of Compound 1 against binding of SNC80 to deltareceptor.

FIG. 5 shows effects of compounds on antinociception and respiration.(A) Effects of Compounds 1 and 2 on antinociception as compared withmorphine. **=p<0.01. (B) Effects of Compounds 1 and 2 on respiratoryminute volume (MV) over a 20-minute period as compared to morphine. *p<0.05, *** p<0.001.

FIG. 6 shows the effects of Compound 2 on antinociception and motorimpairment. (A) The effects of Compound 2 (filled triangles) andmorphine sulfate (MS, filled squares) on antinociception were measuredby the tail flick (TF) test. Also, the effects of Compound 2 (opentriangles) and morphine sulfate (open squares) on motor behavior weremeasured. (*=p<0.05). (B) The bar graph shows the ratio of the areaunder the curve (AUC) for percent motor impairment relative to the AUCfor percent antinociception. This ratio is significantly greater(*p<0.05) for morphine than for Compound 2, consistent with greatermotor impairment relative to analgesia for morphine.

FIG. 7 shows the effects of compounds on drug abuse liability. (A) Theeffects of Compound 1 (filled triangles), morphine (filled squares), andvehicle (filled circles) on antinociception were measured by the tailflick (TF) test. * p<0.05. (B) The cumulative doses of either morphineor Compound 1 that were shown to produce maximal antinociception asshown in (A) were tested for the ability to induce conditioned placepreference (CPP). * ** p<0.01.

FIG. 8 shows the duration and relative potency of compounds in reversingchronic pain induced by nerve injury (neuropathic pain). (A) Thedecrease in paw pressure required for withdrawal after nerve injurysurgery was reversed by morphine and Compounds 1, 2, and 5 (squares,down triangles, diamonds, and up triangles, respectively). Times atwhich the reversal was significantly above vehicle (p<0.05 to 0.001) areshown in bars at the top. Scores for Compound 1 were also significantlyabove those of morphine from 155 to 215 min (dashed bar). Compound 5showed similar reversal (80 min) relative to morphine, and Compounds 1and 2 showed significantly longer reversal (120 and 260 min,respectively) relative to morphine. (B) Dose-response curves show thatall three analogs are significantly more potent than morphine, asdetermined by the dose required to fully (100%) reverse hyperalgesia(pre-surgical minus post-surgical pressure).

FIG. 9 shows the extent of tolerance produced by intrathecal delivery ofmorphine or Compound 2 for 1 week via an osmotic minipump. Cumulativedose-response curves (four increasing quarter-log doses) were used andresponses expressed as % maximum possible effect (% MPE) in a tail-flicktest were determined before and after implantation of a minipump. Theshift in ED₅₀ after Compound 2 (about 8.5-fold) was significantly lessthan that after morphine (64 fold), consistent with reduced induction oftolerance by the analog. Similar results were observed with Compounds 1and 5.

FIG. 10 shows activation of glia after 1 week of treatment with morphinebut not analogs. Integrated density of GFAP (A) and pp38 (B) staining inmorphine-treated, but not analog-treated rats, is significantlyincreased relative to those given vehicle. In addition, the density ofstaining after morphine is significantly greater than that after analogs(*, **, ***=p<0.05, 0.01, 0.001, respectively; n=5-7).

FIG. 11 shows EM analog structures and receptor selectivity. (A)Chemical structures of cyclized, D-amino acid-containing analogs:Tyr-c[D-Lys-Trp-Phe-Glu]-NH₂ (SEQ ID NO: 1, ZH850, Analog 1),Tyr-c[D-Glu-Phe-Phe-Lys]-NH₂ (SEQ ID NO: 2, ZH831, Analog 2),Tyr-c-[D-Lys-Trp-Phe-Asp]-NH₂ (SEQ ID NO: 5, ZH809, Analog 3) andTyr-c[D-Lys-Trp-Phe-Glu]-Gly-NH₂ (SEQ ID NO: 4, ZH853, Analog 4). (B) Invitro opioid receptor selectivity of binding and activation by analogsand reference compounds morphine, DAMGO and EMs to cloned human mu,delta and kappa opioid receptors. Values represent means of 2-4independent assays in duplicate. (C) In vivo selectivity illustrated byantagonism of analog-induced antinociception by naloxone, but not bydelta (NTI) or kappa (nBNI) antagonists. AUC was calculated fromantinociceptive % MPE scores. To provide similar AUC values, Analogs 1and 2 were given at 3.2 mg/kg while Analogs 3 and 4 were given at 5.6and 1.8 mg/kg respectively. Differences among treatments (F_(3,18)=8.30,F_(3,17)=9.34, F_(3,16)=17.33, F_(3,20)=18.93, p<0.001 for Analogs 1-4,respectively) were attributable to a highly significant reduction ofantinociception by naloxone (***p<0.001), while NTI and nBNI treatmentdid not affect antinociceptive scores (n=5-7).

FIG. 12 demonstrates Analog 4 stability and antinociceptiveeffectiveness after peripheral administration. (A, B): Analog 4 wasincubated at 37° C. in human (A) or rat (not shown) plasma orphysiological saline (B) and stability was analyzed at various timepoints by HPLC. Means±SEM of duplicate samples at each point areplotted. Dashed lines represent the 50% degradation point. Linearregressions were significant for plasma and saline (F_(1,32) =216,F_(1,26)=242, p<0.0001) with R² of 0.87 and 0.86, respectively. (C-E):Dose-dependent TF antinociception after i.v., s.c., and oraladministration of Analog 4. Two-way ANOVA showed a significant effect ofdose (F_(3,276)=252.4, F_(4,310)=76.00, F_(3,182)=117.00) time(F_(12,276)=17.7, F_(10,310)=20.4, F_(10,182)=26.5) and interaction(F_(36,276)=4.3, F_(40,310)=4.1, F_(30,182)=4.8), all p<0.0001 for i.v.,s.c., and oral, respectively. (D) The MOR antagonist β-FNA blocked theeffects of Analog 4 (3.2 mg/kg, s.c. [F_(2,192)=53.51, p<0.0001]). Timepoints at which Analog 4 produced significant differences from vehicleare shown at the top of each graph (**,***,**** =p<0.01, 0.001, 0.0001for highest dose vs vehicle; +,++,+++,++++=p<0.01, 0.001, 0.0001 formiddle dose vs vehicle, #=p<0.05 for lowest dose vs. vehicle. n=5-11).

FIG. 13 illustrates reduced impairment of respiration and motorcoordination with BBB penetration. (A) Time course of change in minuteventilation (MV) after morphine (5.6, 10 mg/kg i.v.) or EM Analog 4 atdoses (3.2, 5.6 mg/kg i.v) producing greater duration ofantinociception. (B) Mean % inhibition over 20 min showed significantdifferences after various drug doses (F_(9,58)=6.19, p<0.0001). (C)Duration of antinociception varied among drug groups (F_(9,65)=8.70,p<0.001) with Analog 4 producing almost twice the duration of morphine.Dashed lines illustrate the range of morphine responses for reference.(D) For motor coordination, morphine (squares), Analog 2 (diamonds) orAnalog 4 (triangles) were administered in cumulative doses increasingevery 20 min followed 15 min later by TF and rotorod tests. % MPE valuesfor antinociception (TF, filled symbols) and % maximum possibleinhibition (% MPI) values for rotorod inhibition (open symbols) weredetermined. Differences in antinociception (F_(3,327) ⁼348.9,F_(15,327)=93.51, p<0.0001, for drug and time, respectively) reflectedsignificantly longer and greater total antinociception for Analogs 2 and4 relative to morphine, and differences in motor impairment(F_(3,231)=15.19, F_(10,231)=2.34, p<0.0001, 0.05, for drug and time,respectively) reflected less total impairment. (E) Significantlydifferent motor impairment AUC/antinociceptive AUC ratios(F_(3,21)=4.43, p<0.05) were found and (F) dose-response curves showedthat, on a molar basis, Analogs 2 and 4 were significantly more potentthan morphine (ED₅₀=3.5±0.39, 3.3±0.13, and 5.2±0.41 μmol for Analog 2,Analog 4, and morphine respectively). (G) Antinociception afterperipheral administration of the analogs was reduced by centraladministration of the opioid antagonist N1x-M (F_(11,43)=23.01,p<0.0001) suggesting blood-brain-barrier penetration. TF data wereconverted to area under the curve (AUC). +, ++, +++,++++p<0.05, 0.01,0.001, 0.0001 relative to vehicle; *,**, ***, ****p<0.05, 0.01, 0.001,0.0001 relative to morphine, n=5-8.

FIG. 14 illustrates reduced tolerance, glial and CGRP activation afterEM analogs relative to morphine. (A) Tail-flick dose-response curveswere determined before (pre, open symbols) and after (post, closedsymbols) 7 days of intrathecal (i.t.) drug delivery, and shifts in ED₅₀were determined as an index of relative tolerance. The acute potenciesof the analogs were 30-fold greater than that of morphine before chronicinfusions. The ED₅₀ of morphine shifted 37.7-fold while those of theanalogs shifted on average 13.5-fold reflecting significantly lesstolerance (F_(1,114)=135, p<0.0001). (B) Photomicrographs ofrepresentative samples of dorsal horn immunostaining for the astrocyticmarker GFAP, the microglial marker Iba1, and the MAPK signaling kinaseassociated with microglial activation pp38 [20x; insets: 63× (GFAP,Iba1) and 40× (pp38), Scale bar=100 μm]. (C) Significant differences inimmunostaining were observed for all three markers (F_(5,25)=3.61,p<0.05, F_(5,24)=4.71, p<0.01, F_(5,25)=16.83, p<0.0001 for GFAP, Iba1and pp38, respectively). All three were activated by morphine, but nonewere activated by the analogs. Values for all analogs were significantlybelow those of morphine for Iba1 and pp38. (D) Representativephotomicrographs (Scale bar=50 μm) of CGRP expression. (E)Quantification showing increased CGRP immunostaining after chronicmorphine, but not analogs (F_(5,24)=4.50, p<0.01). CGRP levels after theanalogs were significantly reduced compared to morphine. (F,G)Upregulation of P2X7 receptors in microglial cells. (F) Representativephotomicrographs (Scale bar=20 μm); and (G) quantification showingchronic morphine upregulated P2X7 receptors (t₇=2.50, p<0.05),OX-42-labeled microglial cells (F_(2,16)=3.96, p<0.05), and especiallymicroglial cells containing P2X7 receptors (merge) (F_(2,12)=15.61,p<0.001). By contrast, Analog 4 did not alter OX-42 levels or microglialco-labeling with P2X7 receptors. +, ++, ++++=p<0.05, 0.01, 0.0001significantly different from vehicle; *,**,*** =p<0.05, 0.01, 0.001,compared to morphine. Tolerance: n=6-12; IHC: n=5-6 rats, 4-6 sectionsper rat.

FIG. 15 demonstrates that chronic morphine, but not Analog 4, inducesthermal hyperalgesia. Tail flick latencies were measured on day 1(baseline, BL) and after 7 day infusion (D7) of morphine or Analog 4.Heat settings for baseline latencies were set to 10 sec to allowassessment of increased thermal sensitivity with a cutoff of 20 sec.Latencies were significantly reduced after 7 day infusion of morphine,but not Analog 4. [+=p<0.05 relative to BL, n=12 (morphine), 8 (Analog4)].

FIG. 16 illustrates tests for abuse liability. (A) Doses of morphine andEM analogs providing equal antinociception 20 min after injection. (B)Conditioned place behavior at equi-antinociceptive doses showed morphineproduced a significant increase in time spent in the drug-paired box(CPP), while rats given EM analogs were not significantly different fromcontrols (F_(5, 51)=3.25, p<0.05). (C) CPP for morphine occurred in aclassic inverted-U dose-response fashion, whereas analogs did notproduce CPP (F_(6,57)=3.97, p<0.01). (D) Self-administration: Ratselevated lever pressings when required to obtain a sub-antinociceptivedose of morphine (F_(1, 18)=20.33, p<0.001), but not for infusions(“inf”) of the analogs, and only Analog 2 showed a small but significantincrease on the active “drug lever” vs. inactive lever on the finaltrial (F_(5, 272)=36.11, p<0.0001). (E) Active (filled bars) andinactive (open bars) lever pressings averaged across sessions 5-7(FR3-5) from Panel D. Active lever pressings for morphine, but notanalogs, were significantly greater than the inactive lever and vehicle(F_(11, 78)=10.16, p<0.0001). (F) A variable dose experiment showed thatas the available dose was lowered, rats worked harder to obtainmorphine, but not analog infusions (F_(4, 240)=32.05,p<0.0001). (G)Lever pressings for antinociceptive doses of morphine were significantlygreater than the inactive lever (morphine 1 mg/kg/inf: F_(1, 55)=18.33,p<0.0001; 3 mg/kg/inf: F_(1, 49)=21.66, p<0.0001), while these doses ofAnalog 4 did not produce self-administrations. (H) Number of infusionsand intake per 12 h shows that Analog 4 was not self-administered at anydose compared to morphine. (I) Active lever pressings show ratsincreased workload effort for morphine infusions at a range of doses(F_(6, 42)=5.395, p<0.001) while no escalation was made for Analog 4(F_(3, 25)=2.05, p=0.1321). +,++,+++p<0.05, 0.01, 0.001 relative tovehicle; *,**,*** p<0.05, 0.01, 0.001 relative to morphine; #, ##p<0.05,0.01 relative to the inactive lever. n=8 rats/group for CPP; n=5-10rats/group SA.

FIG. 17 shows that EM analogs provide potent and prolonged relief ofneuropathic pain induced by the spared nerve injury (SNI) model in therat. As demonstrated in Panel A, prior to SNI surgery (“pre-surgery”),an average pressure of about 182 g applied to the hindpaw with aRandall-Selitto device was required to elicit a paw withdrawal response.At 7 to 10 days post-surgery, the animals showed hyperalgesia, indicatedby a reduction in the average pressure (to about 74 g) required toelicit withdrawal. Drugs were administered as intrathecal cumulativedoses chosen to produce full alleviation of the hyperalgesia. Pressuretolerance (pain alleviation) was significantly greater than vehicleuntil 135 min after injection for morphine, and 235 min or more for theanalogs (p<0.05−0.0001). In addition, scores for analog 2 weresignificantly above those of morphine at 135 min, from 135-235 min forAnalog 1, and from 95-235 min for Analog 4 (bars above time points,p<0.05-0.0001). ‘ns vs veh’=time at which morphine scores were no longersignificantly different than vehicle. Dose-response curves (Panel B)showed that all analogs are significantly more potent than morphine, asdetermined by the dose required to fully (100%) reverse thehyperalgesia, i.e., return to the pre-surgical baseline response(pre-surgical minus post-surgical pressure). While potency differencesamong the analogs were not significant (p>0.05), the analogs reversedmechanical hypersensitivity at doses about 80-fold lower than morphine(average of 0.02 vs 1.61 μg for morphine, p<0.0001), n=6-11.

FIG. 18 illustrates the antinociceptive effects of several hexapeptidesof the Formula I in which the L-amino acid (AA) in X₅ is varied.Antinociception was measured in the tail flick test followingsubcutaneous injection to mice. All compounds were compared at 3.2 and5.6 mg/kg; results from 9 peptides with X₂=Trp are shown, along with thelisted amino acid substituted in position 6 (X₅). The Gly-containingpeptide (Analog 4, SEQ ID NO: 3) provided potent antinociception afterperipheral injection. Panel A shows the time course of antinociceptioninduced by Analog 4, expressed as maximum possible effect (% MPE, asdescribed for FIG. 6). The 5.6 mg/kg dose maintainedantinociception >50% MPE for 80 min. The area under the curve (AUC) forthe 3.2 and 5.6 mg/kg doses are plotted in Panel B along with those ofthe other analogs. Two peptides (dark bars) produced potentantinociception similar to the Analog 4, i.e., a peptide withsubstitution of a basic AA (Arg) in position 6, and a peptide withsubstitution of 2,6-dimethyl-L-tyrosine (DMT) in place of Tyr inposition one (DMT¹-Gly). Significant reduction of potency was observedby substitution of Tyr, Met, and Pro in position 6 (white bars),relative to Analog 4, although the compounds were still active, whileLeu, Gln, and Ser showed intermediate potency (grey bars). Somepreference for smaller moieties within a class is indicated by thegreater potency of Ser vs Tyr for hydroxyl AAs, and for Gly vs Leu foraliphatic AAs. Positive charge appears preferable to negative charge asreflected in the greater potency of Arg vs Gln. Panel C: In addition toTrp³ hexapeptides, a Phe³ Gly⁶ hexapeptide was compared to the referenceTrp³-Gly⁶ peptide (SEQ ID NO: 3). For this comparison, HCl salt forms ofthe peptides were tested while those in A and B were acetate salts. Theanalogs with an HCl (C) salt form provided a greater area under thecurve relative to acetate salt (A,B), and the Phe³ analog was fullyeffective (C); *,*** =p<0.05, 0.001 main effect of drug relative to Gly⁶analog, n=5. The results indicate that both Tyr and DMT are effective inposition 1, preferential amino acids in position 6 are Gly and Arg, andthat to a lesser extent, several additional amino acids in position 6can induce antinociception.

DETAILED DESCRIPTION OF THE INVENTION

Peptides of Formula I, which are cyclic pentapeptide and hexapeptideanalogs of endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂, SEQ ID NO:8) andendomorphin-2 (Tyr-Pro-Phe-Phe-NH₂, SEQ ID NO:9) were prepared. In eachcase, the cyclic portion of the peptide is formed from amino acidresidues 2 through 4, while the Tyr residue (residue 1) is attached toresidue 2 as a branch. Non-limiting examples of peptides with thecomposition of Formula I include Compounds 1-7 below, wherein the sidechains of amino acid residues 2 (X₁) and 5 (X₄) in the sequence arelinked by an amide bond between the side-chains thereof. The formulae ofCompounds 1, 2, 3, 4, 5, 6, and 7 are shown in Table 1.

TABLE 1 Comound H-Tyr- X₁- X₂- X₃- X₄- X₅ SEQ ID NO: 1 Tyr- c[D-Lys TrpPhe Glu] NH₂  (SEQ ID NO: 1) 2 Tyr- c[D-Glu Phe Phe Lys] NH₂(SEQ ID NO: 2) 3 Tyr- c[D-Lys Trp Phe Glu] Gly-NH₂ (SEQ ID NO: 3) 4 Tyr-c[D-Glu Phe Phe Lys] Gly-NH₂ (SEQ ID NO: 4) 5 Tyr- c[D-Lys Trp Phe Asp]NH₂ (SEQ ID NO: 5) 6 Tyr- c[D-Glu N-Me-Phe Phe Lys] NH₂ (SEQ ID NO: 6) 7Tyr- c[D-Orn Phe p-Cl-Phe Asp] Val-NH₂ (SEQ ID NO: 7)

In some embodiments, the peptides of Formula I includes peptides with anN-alkylated phenylalanine in position 3 (X₂). Alkyl groups suitable inthe peptides of the present invention include (C₁ to C₁₀) alkyl groups,preferably (C₁ to C₆) alkyl groups (e.g., methyl or ethyl). Compound 6illustrates a cyclic analog whose linear primary amino acid sequencecontains an N-methylated phenylalanine in position 3. Other peptides ofthis invention include compounds wherein the amino acid at position 4(X₃) is p-Y-phenylalanine, wherein Y is NO₂, F, Cl or Br, in order toenhance receptor binding and potency. An exemplary peptide (Compound 7),whose linear primary amino acid sequence is provided in SEQ ID NO:7, hasa p-chlorophenylalanine (p-Cl-Phe) in position 4.

Compounds 1 (FIG. 1), 2 (FIG. 2), 5 and 6 are examples of cyclicpentapeptides, and Compounds 3, 4 (FIG. 3) and 7 are examples of cyclichexapeptides of the instant invention.

For reference, the abbreviations for amino acids described hereininclude alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid(Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine(Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys),methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser),threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Val),ornithine (Orn), naphthylalanine (Nal), 2,3-diaminopropionic acid (Dpr),and 2,4-diaminobutyric acid (Dab). The L- or D-enantiomeric forms ofthese and other amino acids can be included in the peptides of FormulaI. Other amino acids, or derivatives or unnatural forms thereof such asthose listed in the 2009/2010 Aldrich Handbook of Fine Chemicals(incorporated herein by reference in its entirety, particularly thosesections therein listing amino acid derivatives and unnatural aminoacids) can be used in preparing compounds of the invention.

In Formula I, X₁ can be, for example, D-Asp, D-Glu, D-Lys, D-Orn, D-Dpror D-Dab, and X₄ can be, for example, Asp, Glu, Lys, Orn, Dpr or Dab. Ingeneral, an amino acid or derivative thereof can be used as X₁ or X₄ ifit contains either an amino group or a carboxyl group in its side chain.In some embodiments, the amino acid used for X₁ can be a D-enantiomericform of such amino acid.

X₂ and X₃ in Formula I are aromatic amino acids. Examples of such aminoacids are unsubstituted or substituted aromatic amino acids selectedfrom the group consisting of phenylalanine, heteroarylalanine,naphthylalanine (Nal), homophenylalanine, histidine, tryptophan,tyrosine, arylglycine, heteroarylglycine, thyroxine, aryl-beta-alanine,and heteroaryl-beta-alanine. Examples of substituted versions of thesearomatic amino acids are disclosed in U.S. Pat. No. 7,629,319, which isherein incorporated by reference in its entirety. As used herein,“aromatic amino acid” refers to an a-amino acid comprising an aromaticgroup (including aromatic hydrocarbon and aromatic heterocyclic groups)in the side-chain thereof.

In some embodiments, X₂ in Formula I can be N-alkyl-Phe, where the alkylgroup comprises 1 to about 6 carbon atoms. Alternatively, the alkylgroup can comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbons, forexample. The alkyl group can be a methyl (i.e., X₂ is N-Me-Phe), ethyl,propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl, isohexyl,heptyl, or isoheptyl group, or any other branched form thereof, forexample. By definition, the alkyl group of N-alkyl-Phe is linked to theα-amino group of phenylalanine. This alpha amino group is involved in anamide bond with the X₁ residue in certain peptides of the invention;therefore, the alpha amino group of X₂ (when N-alkyl-Phe) as it existsin such peptides is a tertiary amide.

In some embodiments X₃ in Formula I is para-Y-Phe (p-Y-Phe), where Y isNO₂, F, Cl, or Br, for example. For example, X₃ can be p-Cl-Phe.Alternatively, the NO₂, F, Cl, or Br groups can be linked in the orthoor meta positions of the phenyl ring of Phe. Any aromatic amino acidincorporated in the compounds of the invention such as at X₂ or X₃ canhave the above groups linked thereto in the ortho, meta, or parapositions.

Solubility.

The solubility of the peptides of Formula I (e.g., in saline orphysiologic buffer) typically is enhanced relative to the prior arttetrapeptide analogs of the endomorphins. Addition of a hydrophilicamino acid and amidated C-terminus to the relatively hydrophobictetrapeptide sequences Tyr-cyclo[D-Lys-Trp-Phe] (SEQ ID NO:10) andTyr-cyclo[D-Lys-Phe-Phe] (SEQ ID NO:11), resulted in an unexpectedlyhigh improvement in solubility while maintaining or improvingfunctionality. For example, Compound 1 was soluble in water, saline and20% PEG/saline at about 43, 21 and 90 mg/mL, respectively, compared toless than about 2 mg/mL for the previously described compounds. Whileincreases in solubility are associated with improved pharmaceuticaldelivery properties, higher solubility is also often associated withreduced functional activity (e.g., receptor binding) that may depend onlipophilicity. Surprisingly however, as described in examples below, thefunctional properties of the compounds of the invention are notdiminished, and indeed are generally improved.

Methods of Preparation of the Peptides of Formula I.

The peptides of Formula I can be prepared by conventional solution phase(2) or solid phase (18) methods with the use of proper protecting groupsand coupling agents; references 2 and 20 are herein incorporated byreference in their entirety. Such methods generally utilize variousprotecting groups on the various amino acid residues of the peptides. Asuitable deprotection method is employed to remove specified or all ofthe protecting groups, including splitting off the resin if solid phasesynthesis is applied. The peptides can be synthesized, for example, asdescribed below.

Peptides of Formula I were synthesized on Rink Amide resin via Fmocchemistry. A t-butyl group was used for Tyr, Glu, Asp side chainprotection and Boc was used for Lys, Orn and Trp side chain protection.All materials were obtained from EMD Biosciences, Inc (San Diego,Calif.). The peptide was assembled on Rink Amide resin by repetitiveremoval of the Fmoc protecting group and coupling of protected aminoacid. HBTU (O-benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate; CAS #94790-37-1) and HOBT (N-hydroxybenzotriazole;CAS #2592-95-2) were used as coupling reagents in N,N-dimethylformamide(DMF) and diisopropylethylamine (DIPEA) was used as a base. The resinwas treated with an aqueous cocktail of trifluoroacetic acid andtriisopropylsilane (TFA/TIS/H₂O cocktail) for cleavage and removal ofthe side chain protecting groups. Crude peptide was precipitated withdiethyl ether and collected by filtration.

Cyclization of the linear Fmoc-Tyr-c[X₁-X₂-X₃-X₄]-X₅ precursors: About 1mmol of peptide was dissolved in about 1000 mL DMF and about 2 mmolDIPEA was added to the solution, followed by a solution of HBTU (about1.1 mmol) and HOBT (about 1.1 mmol) in about 100 mL DMF. The reactionmixture was stirred at room temperature overnight. Solvent was removedin vacuo. The resulting solid residue was washed with 5% citric acid,saturated NaCl, saturated NaHCO₃, and water. The final solid was washedwith diethyl ether and dried under high vacuum.

Preparation of Tyr-c[X₁-X₂-X₃-X₄]-X₅ peptides. The solids obtained abovewere dissolved in 20% piperidine/DMF. The mixture was stirred at roomtemperature for about 1 hour. Solvent was removed in vacuo. Residueswere dissolved in 10% aqueous acetonitrile (MeCN/H₂O) and lyophilized.

Purification of the crude lyophilized peptides was performed withreverse phase high performance liquid chromatography (RP-HPLC). The HPLCsystem GOLD 32 KARAT (Beckman) consisting of the programmable solventmodule 126 and the diode array detector module 168 was used in thepurification and the purity control of the peptides. Reverse phase HPLCwas performed using a gradient made from two solvents: (A) 0.1% TFA inwater and (B) 0.1% TFA in acetonitrile. For preparative runs, a VYDAC218TP510 column (250×10 mm; Alltech Associates, Inc.) was used with agradient of 5-20% solvent B in solvent A over a period of 10 min, 20-25%B over a period of 30 minutes, 25-80% B over a period of 1 minute andisocratic elution over 9 minutes at a flow rate of about 4 mL/min,absorptions being measured at both 214 and 280 nm. The same gradient wasused for analytical runs on a VYDAC 218TP54 column (250×4.6 mm) at aflow rate of about 1 mL/min.

Pharmaceutical Preparations.

The instant invention also provides pharmaceutical preparations whichcontain a pharmaceutically effective amount of the peptides in apharmaceutically acceptable carrier (e.g., a diluent, complexing agent,additive, excipient, adjuvant and the like). The peptide can be presentfor example in a salt form, a micro-crystal form, a nano-crystal form, aco-crystal form, a nanoparticle form, a mirocparticle form, or anamphiphilic form. Salt forms can be, e.g., salts of inorganic acids suchas hydrochloride salts, phosphate salts, sulfate salts, bisulfate salts,hemisulfate salts, and the like; or salts of organic acids, such asacetate salts, aspartate salts, citrate salts, fumarate salts, maleatesalts, malate salts, lactate salts, hippurate salts, tartrate salts,gluconate salts, succinate salts, and the like. The carrier can be anorganic or inorganic carrier, or a combination thereof, which issuitable for external, enteral or parenteral applications. The peptidesof the present invention can be compounded, for example, with the usualnon-toxic, pharmaceutically acceptable carriers for tablets, pellets,capsules, liposomes, suppositories, intranasal sprays, solutions,emulsions, suspensions, aerosols, targeted chemical delivery systems(15), and any other form suitable for use. Non-limiting examples ofcarriers that can be used include water, glucose, lactose, gum acacia,gelatin, mannitol, starch paste, magnesium trisilicate, talc, cornstarch, keratin, colloidal silica, potato starch, urea and othercarriers suitable for use in manufacturing preparations, in solid,semisolid, liquid or aerosol form. In addition auxiliary, stabilizing,thickening and coloring agents and perfumes can be used.

In another aspect, pharmaceutical compositions useful for treating painand related conditions utilizing the compounds of Formula I aredescribed herein. The pharmaceutical compositions comprise at least onepeptide of Formula I in combination with a pharmaceutically acceptablecarrier, vehicle, or diluent, such as an aqueous buffer at aphysiologically acceptable pH (e.g., pH 7 to 8.5), a polymer-basednanoparticle vehicle, a liposome, and the like. The pharmaceuticalcompositions can be delivered in any suitable dosage form, such as aliquid, gel, solid, cream, or paste dosage form. In one embodiment, thecompositions can be adapted to give sustained release of the peptide.

In some embodiments, the pharmaceutical compositions include, but arenot limited to, those forms suitable for oral, rectal, nasal, topical,(including buccal and sublingual), transdermal, vaginal, parenteral(including intramuscular, subcutaneous, and intravenous), spinal(epidural, intrathecal), and central (intracerebroventricular)administration. The compositions can, where appropriate, be convenientlyprovided in discrete dosage units. The pharmaceutical compositions ofthe invention can be prepared by any of the methods well known in thepharmaceutical arts. Some preferred modes of administration includeintravenous (iv), topical, subcutaneous, oral and spinal.

Pharmaceutical formulations suitable for oral administration includecapsules, cachets, or tablets, each containing a predetermined amount ofone or more of the peptides, as a powder or granules. In anotherembodiment, the oral composition is a solution, a suspension, or anemulsion. Alternatively, the peptides can be provided as a bolus,electuary, or paste. Tablets and capsules for oral administration cancontain conventional excipients such as binding agents, fillers,lubricants, disintegrants, colorants, flavoring agents, preservatives,or wetting agents. The tablets can be coated according to methods wellknown in the art, if desired. Oral liquid preparations include, forexample, aqueous or oily suspensions, solutions, emulsions, syrups, orelixirs. Alternatively, the compositions can be provided as a dryproduct for constitution with water or another suitable vehicle beforeuse. Such liquid preparations can contain conventional additives such assuspending agents, emulsifying agents, non-aqueous vehicles (which mayinclude edible oils), preservatives, and the like. The additives,excipients, and the like typically will be included in the compositionsfor oral administration within a range of concentrations suitable fortheir intended use or function in the composition, and which are wellknown in the pharmaceutical formulation art. The peptides of the presentinvention will be included in the compositions within a therapeuticallyuseful and effective concentration range, as determined by routinemethods that are well known in the medical and pharmaceutical arts. Forexample, a typical composition can include one or more of the peptidesat a concentration in the range of at least about 0.01 nanomolar toabout 1 molar, preferably at least about 1 nanomolar to about 100millimolar.

Pharmaceutical compositions for parenteral, spinal, or centraladministration (e.g. by bolus injection or continuous infusion) orinjection into amniotic fluid can be provided in unit dose form inampoules, pre-filled syringes, small volume infusion, or in multi-dosecontainers, and preferably include an added preservative. Thecompositions for parenteral administration can be suspensions,solutions, or emulsions, and can contain excipients such as suspendingagents, stabilizing agents, and dispersing agents. Alternatively, thepeptides can be provided in powder form, obtained by aseptic isolationof sterile solid or by lyophilization from solution, for constitutionwith a suitable vehicle, e.g. sterile, pyrogen-free water, before use.The additives, excipients, and the like typically will be included inthe compositions for parenteral administration within a range ofconcentrations suitable for their intended use or function in thecomposition, and which are well known in the pharmaceutical formulationart. The peptides of the present invention will be included in thecompositions within a therapeutically useful and effective concentrationrange, as determined by routine methods that are well known in themedical and pharmaceutical arts. For example, a typical composition caninclude one or more of the peptides at a concentration in the range ofat least about 0.01 nanomolar to about 100 millimolar, preferably atleast about 1 nanomolar to about 10 millimolar.

Pharmaceutical compositions for topical administration of the peptidesto the epidermis (mucosal or cutaneous surfaces) can be formulated asointments, creams, lotions, gels, or as a transdermal patch. Suchtransdermal patches can contain penetration enhancers such as linalool,carvacrol, thymol, citral, menthol, t-anethole, and the like. Ointmentsand creams can, for example, include an aqueous or oily base with theaddition of suitable thickening agents, gelling agents, colorants, andthe like. Lotions and creams can include an aqueous or oily base andtypically also contain one or more emulsifying agents, stabilizingagents, dispersing agents, suspending agents, thickening agents,coloring agents, and the like. Gels preferably include an aqueouscarrier base and include a gelling agent such as cross-linkedpolyacrylic acid polymer, a derivatized polysaccharide (e.g.,carboxymethyl cellulose), and the like. The additives, excipients, andthe like typically will be included in the compositions for topicaladministration to the epidermis within a range of concentrationssuitable for their intended use or function in the composition, andwhich are well known in the pharmaceutical formulation art. The peptidesof the present invention will be included in the compositions within atherapeutically useful and effective concentration range, as determinedby routine methods that are well known in the medical and pharmaceuticalarts. For example, a typical composition can include one or more of thepeptides at a concentration in the range of at least about 0.01nanomolar to about 1 molar, preferably at least about 1 nanomolar toabout 100 millimolar.

Pharmaceutical compositions suitable for topical administration in themouth (e.g., buccal or sublingual administration) include lozengescomprising the peptide in a flavored base, such as sucrose, acacia, ortragacanth; pastilles comprising the peptide in an inert base such asgelatin and glycerin or sucrose and acacia; and mouthwashes comprisingthe active ingredient in a suitable liquid carrier. The pharmaceuticalcompositions for topical administration in the mouth can includepenetration enhancing agents, if desired. The additives, excipients, andthe like typically will be included in the compositions of topical oraladministration within a range of concentrations suitable for theirintended use or function in the composition, and which are well known inthe pharmaceutical formulation art. The peptides of the presentinvention will be included in the compositions within a therapeuticallyuseful and effective concentration range, as determined by routinemethods that are well known in the medical and pharmaceutical arts. Forexample, a typical composition can include one or more of the peptidesat a concentration in the range of at least about 0.01 nanomolar toabout 1 molar, preferably at least about 1 nanomolar to about 100millimolar.

A pharmaceutical composition suitable for rectal administrationcomprises a peptide of the present invention in combination with a solidor semisolid (e.g., cream or paste) carrier or vehicle. For example,such rectal compositions can be provided as unit dose suppositories.Suitable carriers or vehicles include cocoa butter and other materialscommonly used in the art. The additives, excipients, and the liketypically will be included in the compositions of rectal administrationwithin a range of concentrations suitable for their intended use orfunction in the composition, and which are well known in thepharmaceutical formulation art. The peptides of the present inventionwill be included in the compositions within a therapeutically useful andeffective concentration range, as determined by routine methods that arewell known in the medical and pharmaceutical arts. For example, atypical composition can include one or more of the peptides at aconcentration in the range of at least about 0.01 nanomolar to about 1molar, preferably at least about 1 nanomolar to about 100 millimolar.

According to one embodiment, pharmaceutical compositions of the presentinvention suitable for vaginal administration are provided as pessaries,tampons, creams, gels, pastes, foams, or sprays containing a peptide ofthe invention in combination with carriers as are known in the art.Alternatively, compositions suitable for vaginal administration can bedelivered in a liquid or solid dosage form. The additives, excipients,and the like typically will be included in the compositions of vaginaladministration within a range of concentrations suitable for theirintended use or function in the composition, and which are well known inthe pharmaceutical formulation art. The peptides of the presentinvention will be included in the compositions within a therapeuticallyuseful and effective concentration range, as determined by routinemethods that are well known in the medical and pharmaceutical arts. Forexample, a typical composition can include one or more of the peptidesat a concentration in the range of at least about 0.01 nanomolar toabout 1 molar, preferably at least about 1 nanomolar to about 100millimolar.

Pharmaceutical compositions suitable for intra-nasal administration arealso encompassed by the present invention. Such intra-nasal compositionscomprise a peptide of the invention in a vehicle and suitableadministration device to deliver a liquid spray, dispersible powder, ordrops. Drops may be formulated with an aqueous or non-aqueous base alsocomprising one or more dispersing agents, solubilizing agents, orsuspending agents. Liquid sprays are conveniently delivered from apressurized pack, an insufflator, a nebulizer, or other convenient meansof delivering an aerosol comprising the peptide. Pressurized packscomprise a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orother suitable gas as is well known in the art. Aerosol dosages can becontrolled by providing a valve to deliver a metered amount of thepeptide. Alternatively, pharmaceutical compositions for administrationby inhalation or insufflation can be provided in the form of a drypowder composition, for example, a powder mix of the peptide and asuitable powder base such as lactose or starch. Such powder compositioncan be provided in unit dosage form, for example, in capsules,cartridges, gelatin packs, or blister packs, from which the powder canbe administered with the aid of an inhalator or insufflator. Theadditives, excipients, and the like typically will be included in thecompositions of intra-nasal administration within a range ofconcentrations suitable for their intended use or function in thecomposition, and which are well known in the pharmaceutical formulationart. The peptides of the present invention will be included in thecompositions within a therapeutically useful and effective concentrationrange, as determined by routine methods that are well known in themedical and pharmaceutical arts. For example, a typical composition caninclude one or more of the peptides at a concentration in the range ofat least about 0.01 nanomolar to about 1 molar, preferably at leastabout 1 nanomolar to about 100 millimolar.

Optionally, the pharmaceutical compositions of the present invention caninclude one or more other therapeutic agent, e.g., as a combinationtherapy. The additional therapeutic agent will be included in thecompositions within a therapeutically useful and effective concentrationrange, as determined by routine methods that are well known in themedical and pharmaceutical arts. The concentration of any particularadditional therapeutic agent may be in the same range as is typical foruse of that agent as a monotherapy, or the concentration may be lowerthan a typical monotherapy concentration if there is a synergy whencombined with a peptide of the present invention.

In another aspect, the present invention provides for the use of thepeptides of Formula I for treatment of pain, treatment of discomfortassociated with gastrointestinal disorders, and treatment of drugdependence. Methods for providing analgesia (alleviating or reducingpain), relief from gastrointestinal disorders such as diarrhea, andtherapy for drug dependence in patients, such as mammals, includinghumans, comprise administering to a patient suffering from one of theaforementioned conditions an effective amount of a peptide of Formula I.Diarrhea may be caused by a number of sources, such as infectiousdisease, cholera, or an effect or side-effect of various drugs ortherapies, including those used for cancer therapy. Preferably, thepeptide is administered parenterally or enterally. The dosage of theeffective amount of the peptides can vary depending upon the age andcondition of each individual patient to be treated. However, suitableunit dosages typically range from about 0.01 to about 100 mg. Forexample, a unit dose can be in the range of about 0.2 mg to about 50 mg.Such a unit dose can be administered more than once a day, e.g., two orthree times a day.

All of the embodiments of the peptides of Formula I can be in the“isolated” state. For example, an “isolated” peptide is one that hasbeen completely or partially purified. In some instances, the isolatedcompound will be part of a greater composition, buffer system or reagentmix. In other circumstances, the isolated peptide may be purified tohomogeneity. A composition may comprise the peptide or compound at alevel of at least about 50, 80, 90, or 95% (on a molar basis or weightbasis) of all the other species that are also present therein. Mixturesof the peptides of Formula I may be used in practicing methods providedby the invention.

Additional embodiments of the current invention are directed towardsmethods of using the peptides of Formula I disclosed herein in medicinalformulations or as therapeutic agents, for example. These methods mayinvolve the use of a single peptide, or multiple peptides in combination(i.e., a mixture). Accordingly, certain embodiments of the invention aredrawn to medicaments comprising the peptides of Formula I, and methodsof manufacturing such medicaments.

As used herein, the terms “reducing,” “inhibiting,” “blocking,”“preventing”, alleviating,” or “relieving” when referring to a compound(e.g., a peptide), mean that the compound brings down the occurrence,severity, size, volume, or associated symptoms of a condition, event, oractivity by at least about 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%,25%, 27.5%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%,or 100% compared to how the condition, event, or activity would normallyexist without application of the compound or a composition comprisingthe compound. The terms “increasing,” “elevating,” “enhancing,”“upregulating”,“improving,” or “activating” when referring to a compoundmean that the compound increases the occurrence or activity of acondition, event, or activity by at least about 7.5%, 10%, 12.5%, 15%,17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 750%, or1000% compared to how the condition, event, or activity would normallyexist without application of the compound or a composition comprisingthe compound.

The following examples are included to demonstrate certain aspects ofthe invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples, which representtechniques known to function well in practicing the invention, can beconsidered to constitute preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific disclosedembodiments and still obtain a like or similar result without departingfrom the spirit and scope of the invention. The examples are providedfor illustration purposes only and are not intended to be limiting.

Example 1 Binding and Activation of Human Opioid Receptors

The peptides of Formula I showed surprisingly high affinity(subnanomolar) for the human mu opioid receptor with selective bindingrelative to the delta and kappa opioid receptors. The compounds weretested in standard binding assays using ³H-DAMGO (tritiated [D-Ala²,N-Me-Phe⁴, Gly-ol]-enkephalin; CAS #78123-71-4), ³H-DPDPE(CAS#88373-73-3), and ³H-U69593 (CAS#96744-75-1) to label mu, delta andkappa receptors, respectively, in membranes from CHO cells expressinghuman cloned receptors. As shown in Table 2, endomorphin-1 (EMI, SEQ IDNO:8) and endomorphin-2 (EM2, SEQ ID NO:9) are the most selectiveendogenous mu agonists previously reported. Analogs based on thesenatural opioids show greater affinity for the mu receptor, albeit withless selectivity. Tetrapeptide endomorphin analogs described earlier(U.S. Pat. No. 5,885,958; ck1, Tyr-c[D-Lys-Trp-Phe] (SEQ ID NO:10); ck2,Tyr-c[D-Lys-Phe-Phe] (SEQ ID NO:11)) are also shown. Peptides of FormulaI, which include an amidated carboxy-terminus (Compounds 1, 2, 5)retained high affinity binding, but increased selectivity for the mureceptor.

TABLE 2 Compound binding to opioid receptors. K_(i) (nM) Selectivity MuDelta Kappa Delta/Mu Kappa/Mu Morphine 0.92 242 56 264 61 DAMGO 0.78 589334 754 429 EM1 2.07 1215 >10000 587 >5000 EM2 1.32 5704 >100004328 >5000 ck1 0.32 28 35 90 111 ck2 0.36 3 12 9 33 Compound 1 0.49 132128 267 260 Compound 2 0.73 69 71 94 98 Compound 5 0.43 140 29 328 67

Receptor Activation: GTPγS Functional Assay.

Functional activation of the three opioid receptors was tested instandard assays in which the non-hydrolysable GTP analog, ³⁵S-GTPγS, wasused to quantify activation of cloned human opioid receptors expressedin cell membranes. FIG. 4, Panel A shows that Compound 1 is a fullefficacy agonist with significantly greater potency than the referencecompound, DAMGO. FIG. 4, Panel B shows that Compound 1 exhibitsunexpected full efficacy as a delta antagonist; i.e., it is able toinhibit the delta activation produced by an ED₈₀ dose of the referencedelta agonist, SNC80 (CAS #156727-74-1). Table 3 shows that all agoniststested are potent activators of the mu receptor, with EC₅₀ (medianeffective concentration) values at low-nanomolar to sub-nanomolarconcentrations. All compounds were found to be full efficacy (>90%)agonists at the mu receptor. The endomorphins and the compounds ofFormula I of the invention show remarkable selectivity for receptoractivation, with delta activation below 50% at concentrations up to 10μM, reflecting selectivity >100000. Compounds 1 and 3, however, showedfull efficacy delta antagonism; Compound 1 exhibited this antagonism ata relatively low concentration.

TABLE 3 Opioid receptor activation by compounds. Agonist Delta EC₅₀ (nM)Selectivity Antagonist mu delta kappa delta/mu kappa/mu IC 50 efficacyMS^(a) 3.90 1245 2404 319 616 DAMGO 1.98 3641 13094 1839 6613 ck1 0.21138 469.51 658 2236 ck2 0.15 7 206.11 44 1374 EM11.82 >100000 >100000 >50000 >50000 4287 100 EM28.44 >100000 >100000 >10000 >10000 30000 88 Comp. 1 0.15 >100000963.79 >500000 6425 105 93 Comp. 2 0.99 >100000 12114.00 >100000 122362750 51 Comp. 5 0.22 >100000 740.34 >400000 3365 557 100 ^(a)morphinesulfate

Receptor Activation: Beta-Arrestin Recruitment.

Beta-arrestin is an intracellular protein that is recruited to the muopioid receptor following activation by agonists. It has been shown toactivate intracellular signaling pathways that in many cases areindependent of well-known G-protein mediated pathways. It has recentlybeen shown that beta-arrestin knockout mice exhibit altered responses tomorphine, including increased analgesia and decreased side effects suchas tolerance, respiratory depression, and constipation (16). Theseresults indicate that the analgesic and side-effects of morphine areseparable by manipulation of cell signaling processes. These findingsalso provide support for the recent concept known variously as“functional selectivity”, “biased agonism”,“agonist directed signaling”and other descriptions. According to this concept, agonists capable ofproducing a different cascade of signaling at a given receptor couldproduce a different profile of desired and undesired effects relative toother agonists for that receptor. Three of the analogs of this inventionwere tested and showed patterns of beta-arrestin recruitment (rangingfrom high potency with low efficacy to moderate potency with significantefficacy) that were different from each other and from morphine.Together with the differential analgesic/side-effect profiles relativeto morphine described in previous examples, the beta arrestin resultssuggest that these compounds exhibit “functional selectivity”, favoringanalgesia over adverse side-effects.

Beyond the value of high mu agonist selectivity (i.e., exclusion ofpotential side-effects resulting from activation of multiple receptors),delta antagonism is expected to attenuate opioid-induced tolerance,dependence, and reward. As first shown in 1991 (1) and supported innumerous studies since, delta antagonists can reduce morphine-inducedtolerance and dependence, while maintaining or enhancing analgesia.Recent studies (11) have also shown reduced rewarding properties of muagonist/delta antagonists as reflected in the conditioned placepreference (CPP) test described below. The activity of the peptides ofFormula I (e.g., Compound 1) as mu agonists/delta antagonists as well asat mu/delta receptor dimers indicate that the peptides will produceeffective analgesia with reduced tolerance, dependence, and reward (18).

Example 2 Providing Analgesia of Greater Duration, but with ReducedRespiratory Depression, Relative to Morphine after IntravenousAdministration

Respiratory depression is a major safety issue in the use of opioids. Anopioid providing analgesia as effective as that produced by morphine,but with less respiratory depression, would be a major advance for thesafe use of opioid analgesics. Effectiveness after systemicadministration, such as intravenous (i.v.) injection, is unusual forpeptide-based compounds, and would be critical for the clinical utilitythereof. Two peptides (Compounds 1 and 2) were tested for their effectson respiration (minute ventilation) and duration of antinociceptionrelative to morphine. Rats with indwelling jugular catheters were placedin a BUXCO whole body plethysmograph apparatus for determining multiplerespiratory parameters. For 20 minutes following i.v. injection ofvehicle (saline), baseline minute ventilation was determined. Animalswere then injected with morphine or test compound and changes frombaseline were determined for 20 minutes, the period of maximalinhibition of minute ventilation by all compounds. The standardtail-flick (TF) test was used to determine antinociception. A baselinetest was conducted before placing the animal in the BUXCO chamber, atthe end of the 20-minute respiratory test, and at every 20 minutesthereafter until the TF latency returned to below 2x baseline TF.Baseline latencies were 3-4 seconds and a cut-off time (“maximalantinociception”) was set at 9 seconds to avoid tissue damage.

FIG. 5, Panel A shows that 10 mg/kg doses of Compounds 1 and 2 producedsignificantly longer antinociception than all other treatments(**=p<0.01) and 5.6 mg/kg doses produced antinociception similar to the10 mg/kg dose of morphine. Despite the greater antinociceptive effect ofCompounds 1 and 2, significantly (* p<0.05) less inhibition ofrespiration was observed in both doses of Compound 1 and in the 5.6mg/kg dose of Compound 2 (FIG. 5, Panel B). These results indicate anunexpected and clearly safer therapeutic profile for the peptides ofFormula I over the current standard opioid analgesic.

Example 3 Providing Analgesia of Greater Duration than Morphine withReduced Impairment of Neuromotor Coordination and Cognitive Function

Neuromotor and cognitive impairment are characteristics of opioids thatare of particular importance in two populations, i.e., military combattroops, where escape from immediate danger can require unimpaired motorand cognitive skills, and the elderly, where these impairments canexacerbate compromised function including impaired balance, which canlead to increased risk of fractures.

Example 3a Neuromotor Coordination

FIG. 6, Panel A illustrates that Compound 2 produces significantlygreater antinociception, but significantly reduced motor impairment,relative to morphine (MS). Both compounds were administered bycumulative intravenous (i.v.) doses in rats. Increasing quarter-logdoses were given every 20 minutes, and a tail flick (TF) test (a test oflatency to remove the tail from a hot light beam) followed by a rotorodtest were conducted about 15 minutes after each injection. Escalatingdoses were given until each animal showed greater than 90% maximumpossible effect (% MPE) on the TF test, determined as: [(latency to TFminus baseline latency)/(9 sec maximum (cut off) time to avoid tissuedamage) minus baseline)]×100. The animal was then placed on a rod thatrotated at speeds escalating to 13 revolutions per minute (RPM) over 3minutes, and the latency to fall from the rod was determined. Onlyanimals that consistently remained on the rod for the full 180 secondsduring training in the drug-naïve state were tested. % Maximum PossibleInhibition (% MPI) of motor coordination was determined as 100−(latencyto fall/180×100).

The two compounds showed similar onset to maximal antinociception, butCompound 2 produced significantly longer antinociception, as reflectedby TF latencies significantly (*=p<0.05) longer than those of themorphine group at 135 and 155 minutes (FIG. 6, Panel A). Despite thisgreater antinociception, the motor impairment was significantly lessthan that of morphine (FIG. 6, Panel B, * p<0.05). The impairment ofmotor behavior by morphine was significantly above that of vehiclecontrols (* p<0.05) while that of Compound 2 was not.

Example 3b Cognitive Impairment

A widely used standard test of cognitive function is the Morris WaterMaze (MWM). During training, rats learn to find a hidden escape platformbased on spatial memory. Average latency to the platform, as well asaverage distance from the platform (a measure unaffected by swim speed),decrease as the task is acquired and provide indices of spatial memory.After 4 days of training, an injection of morphine produced impairmentof spatial memory, as reflected by a significant increase in the latencyto, and average distance from, the platform. By contrast, Compound 2, atdoses that provide equal or greater antinociception than morphine, didnot produce significant impairment. These results indicate an unexpectedand superior therapeutic profile of the peptides of Formula I withregard to cognitive function relative to the current standard opioidanalgesic.

Example 4 Providing Analgesia of Greater Duration, but Reduced Reward,Relative to Morphine

Opioids remain the standard treatment for relief of severe pain, butdiversion of pain medications for non-pain use has become a seriousnational problem (see U.S. Department of Health and Human ServicesSubstance Abuse and Mental Health Services Administration, found atworld wide websiteoas(dot)samhsa(dot)gov/2k9/painRelievers/nonmedicalTrends(dot)pdf).Considerable efforts in academia and industry have focused on“tamper-proof” versions of opioid medications, but there has been littlesuccess in developing opioids that provide highly effective analgesiawith minimal abuse potential. The conditioned place preference (CPP)paradigm is a widely accepted model for demonstrating rewardingproperties of drugs, and all major classes of abused drugs produce CPP,including opioids such as morphine and heroin. Briefly, animals arefirst allowed, on Day 1, to freely explore a 3-compartment apparatusconsisting of a small “start box” and two larger compartments that areperceptually distinct (gray vs. black and white stripes in thisexample). For the next three days, the animals are given an i.v.injection of drug and confined to one compartment, and vehicle is givenin the other. The time at which the drug or vehicle is given (a.m. orp.m.) is counterbalanced, as is the compartment in which the drug isgiven (preferred or non-preferred, as determined during the baselinetest). This unbiased design allows for detection of both drug preferenceand drug aversion. After three days of conditioning (Days 2, 3 and 4),the animal is allowed free access to all compartments on Day 5 in thedrug-free state and the change in absolute time and proportion of timespent in the drug-paired compartment are determined. A significantincrease in the time or proportion of time spent in the drug-pairedcompartment on the post-conditioning test day relative to that on thepre-conditioning baseline test is interpreted as a conditioned placepreference, reflective of rewarding properties and potential abuseliability.

When the cumulative doses of either morphine or Compound 1 that wereshown to produce maximal antinociception (FIG. 7, Panel A) were testedfor the ability to induce CPP (FIG. 7, Panel B), morphine produced asignificant (*** p<0.01) increase in the time spent on the drug side,while Compound 1 did not, even though significantly (* p<0.05) greaterantinociception (FIG. 7, Panel A) was observed with Compound 1 fromabout 140 to 180 minutes after its injection. Compounds 2 and 5 alsoshowed no significant CPP at doses producing antinociception equal tothose of morphine that produced CPP. In a complementary paradigm inwhich rats were provided access to morphine or EM analogs forself-administration, access to morphine, but not analogs, resulted insignificant self-administration. These findings are consistent with lessabuse liability for the novel analogs relative to morphine.

Example 5 Alleviation of Chronic Pain

Chronic pain affects a large proportion of the population. One form ofchronic pain, neuropathic pain, is particularly difficult to treat. FIG.8 shows that Compounds 1, 2 and 5 provide unexpectedly potent relief ofneuropathic pain induced by the spared nerve injury (SNI) model in therat. As demonstrated in FIG. 8, Panel A, prior to SNI surgery(“pre-surgery”), an average pressure of about 177 g applied to thehindpaw with a Randall-Selitto device was required to elicit a pawwithdrawal response. About 7 to 10 days post-surgery, the animals showedhyperalgesia, indicated by a reduction in the average pressure (to about70 g) required to elicit withdrawal. Drugs were administered asintrathecal cumulative doses chosen to produce full alleviation of thehyperalgesia. Times at which the reversal was significantly (p<0.05 to0.001) above vehicle are shown in bars at the top. Compound 5 showedsimilar reversal times (about 80 min), and Compounds 1 and 2 showedsignificantly longer reversal times (about 120 and 260 min,respectively) relative to morphine (about 80 min). Scores for Compound 1were also significantly above those of morphine from 155-215 min (dashedbar). Dose-response curves (FIG. 8, Panel B) showed that all threeanalogs are significantly more potent than morphine, as determined bythe dose required to fully (100%) reverse the hyperalgesia, i.e., returnto the pre-surgical baseline response (pre-surgical minus post-surgicalpressure). Compounds 1, 2, and 5 reversed mechanical hypersensitivity atdoses about 80-fold to 100-fold lower than morphine (about 0.01 to 0.014μg compared to about 1.14 μg for morphine). On a molar basis, thisrepresents about 180 to 240 fold greater potency than morphine againstneuropathic pain. Similar results were observed after other forms ofchronic pain including post-incisional (post-operative) and inflammatorypain induced by Complete Freund's Adjuvant (CFA). The foregoing examplesare illustrative, but not exhaustive, as to the types of acute orchronic pain for which the peptides of Formula I are effective.

Example 6 Reduced Tolerance and Glial Activation Relative to Morphine

A major limiting factor for the usefulness of opioid medications istolerance, which requires increasing doses to maintain an analgesiceffect. Reduction of the potential for tolerance would be a veryimportant advantage for a novel analgesic. In addition, several recentstudies have shown that repeated opioid exposure sometimes leads to“paradoxical” opioid-induced pain. Increased responsiveness to normallynoxious stimuli (hyperalgesia) or normally non-noxious stimuli such astouch (allodynia) have been reported. Explanations for the tolerance andopioid induced hypersensitivity include the possibility that activationof glia, a reflection of an inflammatory response, results in anincreased release of substances that activate or sensitize neuronaltransmission of nociceptive signals. Specifically, enhanced release of“pronociceptive” cytokines and chemokines are thought to mediate theenhanced pain sensitivity sometimes observed after chronic exposure toopioids. In addition, several studies have linked this phenomenon toopioid tolerance based on the concept that increasing doses of opioidsare required to overcome the increased pronociceptive effects of thereleased compounds. Described below are the unexpected findings that:(1) Compounds 1, 2 and 5 produce significantly less tolerance relativethan morphine, and (2) that in direct comparison to morphine, and incontrast to morphine and most clinically used opioids, the analogs donot induce an inflammatory glial activation response after chronicadministration. In addition to their potential value for reducedescalation of doses required during chronic administration, the analogsof Formula I could be ideal for opioid rotation and for a wide range ofsituations where ongoing inflammatory conditions may be exacerbated bytreatment with morphine. This approach would also be superior to use ofan anti-inflammatory agent as an adjuvant to opioid treatment.

Compounds 1, 2 and 5 all showed greater potency, reduced tolerance andreduced glial activation relative to morphine. For simplicity, onlyCompound 2 is shown in comparison to morphine in FIG. 9. The experimentwas designed to model clinical use of opioids by titrating to fullantinociception in each subject, and maintaining steady blood levels, inthis case through use of osmotic minipumps. Doses producing matchedinitial antinociception were determined for morphine and analog byintrathecal injection of the cumulative dosing paradigm described abovefor the rotorod and neuropathic pain models. Doses were increased untileach rat achieved full antinociception (100% MPE). The ED₅₀ for allcompounds in opioid naïve animals was determined and Compound 2 wasfound to be over 20-fold more potent (p<0.001) than morphine (ED₅₀=0.01μg±0.001 compared to 0.253 μg±0.05 for morphine, n=5-7). This translateson a molar basis to about 40-fold greater potency for the analog.Immediately after the first test, ALZET osmotic minipumps (Durect Corp,Cupertino, Calif.) were implanted subcutaneously and connected to theintrathecal catheter. The primed pumps delivered morphine or analog at 2μg/hr or 0.056 μg/hr for about 7 days, respectively. The 2 μg/hrmorphine dose was chosen based on previous studies in which this dosewas shown to produce glial activation in the dorsal horn in a similarparadigm (19). The dose of analog was chosen using a similar ratio tothe ED₅₀ (about 7× to 8×). A second cumulative dose-response curve wasgenerated on Day 7 after minipump implantation to determine the shift inED₅₀ as an index of relative tolerance. As shown in FIG. 9, the ED₅₀ ofmorphine shifted to 16±3.3 μg (over 60-fold) while that of compound 2shifted only about 8.5 fold to about 0.11±0.02 μg. Compounds 1 and 5showed similar results with potencies over 20×greater than morphine andshifts less than 20 fold. These results show that EM analogs causeunexpected and significantly less tolerance than morphine.

As shown in FIG. 10, morphine produced significant glial activation, butfor all 3 analogs, activation was not significantly different fromvehicle and was significantly less than morphine, establishingdifferential glial effects for morphine compared to EM analogs(Compounds, 1, 2, and 5). Rats used in the above tolerance experimentwere perfused after the final behavioral test and analyzed for glialactivation as indicated by (A) GFAP staining for astroglia and (B)phospho-p38, a signaling pathway activated in microglia by morphine.Five sections from each of 5-7 animals/group were analyzed forintegrated density of staining with the IMAGE J program. Morphine, butnone of the analogs, showed significantly greater induction thanvehicle. Values for all analogs were significantly below those ofmorphine (*,**,***=p<0.05, 0.01, 0.001, respectively, compared toindicated groups). These data provide evidence that, at doses producingequal or greater antinociception, the analogs produce unexpectedly lessglial activation and this is associated with reduced tolerance.

Example 7 Evaluation of a Hexapeptide Analog of Formula I Relative toPentapeptides

Chemicals:

A series of pentapeptide and hexapeptide compounds of Formula I, i.e.,Compounds 1 (SEQ ID NO: 1), 2 (SEQ ID NO; 2), 5 (SEQ ID NO: 5), and 4(SEQ ID NO: 4) described above (referred to hereinafter in this Exampleas Analog 1, Analog 2, Analog 3, and Analog 4, respectively) weresynthesized by standard solid phase methods at 1 mMol on a Rink amideresin via fluorenylmethyloxycarbonyl (Fmoc) chemistry with purity (>95%)and sequence identity confirmed by HPLC and MS. Analogs selected forfull characterization (FIG. 11, Panel A) were synthesized at 2 g scale.Morphine sulfate and beta-funaltrexamine (β-FNA) were supplied by NIDA,naloxone and naloxone methiodide (Nlx-M) were obtained from Sigma (St.Louis, Mo.), naltrindole (NTI) and nor-binaltorphimine (nBNI) fromTocris (Ellisville, Mo.), and Tyr-D-Ala²-N-MePhe⁴-Gly-ol (DAMGO) fromBachem (King of Prussia, Pa.).

Receptor binding assays were conducted using cloned human receptors inCHO-K1 cell membranes and the following ligands for mu, delta, and kappaopioid receptors (MOR, DOR KOR), respectively: ³H-DAMGO, ³H-DPDPE and³H-U69593. Membranes, radioligands at their Kd concentrations, andvarying concentrations of test compounds were incubated in 50 mM Tris pH7.4, 5 mM MgCl₂, 10 μg/mL saponin, for 60 min at 25° C., filtered overGF/B filters and counted in MICROSCINT 20 scintillation cocktail(Packard).

Activation of receptors was determined in GTPγS assays. CHO-K1 cellmembranes, as described above, were mixed with GDP (1 μM for MOR, 10 μMfor DOR and KOR) for 15 min on ice followed by incubation with ³⁵5-GTPγS(0.1 nM) and scintillation proximity assay (SPA) beads in 20 mM HEPES(pH 7.4) containing 10 mM saponin, MgCl₂ (1 mM for MOR and DOR, 30 mMfor KOR) and 100 mM NaCl. After shaking for 2 min and centrifugation(2000 rpm, 10 min), samples were incubated for 1 hour (hr) and counted.Percent efficacy was calculated relative to activation by referencecompounds DAMGO, SNC80, and U-50488 for mu, delta and kappa receptors.All test compounds showed >95% efficacy at MOR. DOR antagonism wasassessed by inhibition of the activation produced by SNC80 at its EC₈₀.

Stability in 37° C. Plasma and Saline.

To test analog degradation by human and rat plasma enzymes in vitro,pooled normal human plasma was obtained from Innovative Research (NoviMich., cat.#IPLA-N) and rat aortic blood was drawn in a 10 ml syringerinsed with heparin, incubated at room temperature 1 hr and centrifuged(2000 g×15′, 4° C.). Analogs were incubated at 200 μg/ml in fresh plasma37° C. Aliquots (75 μL=7.5 μg) of the mixture were withdrawn at varioustimes and immediately added to 75 μL ice cold 0.1M HCl, and centrifugedat 30,000 g, 20 min, 4° C. Aliquots (100 μL, about 5 μg) of thesupernatant were frozen pending HPLC analysis. Samples were diluted with100 μL 0.1% TFA in water and analyzed on a Beckman System GOLD HPLC witha VYDAC 218TP54 C18 column using eluents comprising 0.1% TFA inwater/acetonitrile (AcN) mixtures. The samples were run at 1 mL/min ingradients of 5-20% AcN/10 min, 20-25% AcN/30 min, 25-60% AcN/1 min, 60%AcN/9 min, and 60-80% AcN/1 min with an absorbance detector andabsorbance was plotted as a function of elution time. Intact peptide wascalculated from area under the absorbance curve at the appropriateretention time relative to a standard curve generated with the sameconditions. Degradation was calculated by linear regression of nlog A/A₀at 280 nm absorbance. To test analog stability as an injectable solutionat physiological temperature, Analog 4 was sterile-filtered andincubated at 37° C. in sterile saline (10 μg/100 μL). Aliquots wereremoved at various times and analyzed by HPLC. Confirmation of peptideMW/integrity was conducted on an Applied Biosystems VOYAGER-DE PRO massspectrometer.

Animals and Surgery:

Male CD-1 mice (22-25 g) and Sprague-Dawley rats (250-400 g, CharlesRiver, Wilmington, Mass.) were housed in a 12-h light/dark cycle. Allexperiments were approved by the Tulane Institutional Animal Care andUse Committee and conducted according to the NIH Guide for the Care andUse of Laboratory Animals. All efforts were made to minimize animalsuffering, and to reduce the number of animals used. No alternatives toin vivo techniques are available. Drug injections to rats were given asdescribed previously through indwelling jugular vein (i.v.) (55) orintrathecal (i.t.) catheters (60). Mice received subcutaneous (s.c.)injections at the nape of the neck or oral administration by gavage.

Antinociception was determined in a standard tail flick (TF) testwherein the latency to withdraw the tail from a heat source wasautomatically measured (IITC, Woodland Hills, Calif.). Baselinelatencies were 3-4 sec with a cutoff time of 9 sec to prevent tissuedamage. Percent Maximum Possible Effect (% MPE) was determined as[(latency-baseline latency)/(9-baseline latency)]*100.Equi-antinociceptive asymptotic doses of morphine and analogs producing≧95 MPE were used in the CPP test. Doses of morphine and Analog 4 weretested at 0.25 log lower than the maximal antinociceptive dose(producing 60-80% MPE), and 0.25 log higher than the maximalantinociceptive dose. Antinociception after a 20 min respiratory testwas scored as duration of analgesia, defined as time that MPE wasgreater than 50%. Bolus injections were used except for the rotorod(i.v.) and tolerance (i.t.) tests, where cumulative dosing was used (44)with minor modifications. Doses were increased in 0.25 log incrementswith injections every 20 min, followed 15 minutes later by TF androtorod tests, or a TF test alone in the tolerance experiment.

Blood-Brain Barrier Penetration:

Intracerebroventricular (icy) administration of the opioid antagonistnaloxone-methiodide (Nlx-M) was used to test the central effects ofperipherally injected analogs. Rats were placed in a stereotaxicapparatus under isoflurane/oxygen anesthetic (4-5% induction, and1.5-2.5% for maintenance). Infusions of Nlx-M (10 μg/5 μL, icy) orvehicle (5 μL, icy) were made to the right lateral ventricle (1.5 mmlateral, 0.7 mm posterior, and 3.5 mm ventral to the bregma) using a 5μL syringe. The 10 μg icy dose of Nlx-M was chosen based on reports(24,33) showing the effectiveness of this icy dose in antagonizingsystemic morphine. Injection was made over 1 min and the syringe washeld in place for 1 additional min to ensure adequate diffusion. About20 min after the icy injection, the analogs were injected (i.v.) and TFlatencies were measured 15, 30, 45, 60, 90, 120, 180, 240, 300, and 360mins after injection.

Respiratory depression was measured in unanesthetized free-moving ratsin a whole body plethysmography system (Buxco, Wilmington, N.C.) aspreviously described (29). Rats were given saline (1 mL/kg) through ani.v. jugular PE-50 catheter connected via swivel spring leash to asyringe outside the chamber. Minute ventilation (MV, tidalvolume×respiratory rate) was determined for 20 min (vehicle baseline),then morphine or an EM analog was injected. Change in MV (% vehiclebaseline) was determined over 20 min, the period of maximal effect. A TFtest was then conducted at 20 min intervals until antinociceptive scoresfell below 50% MPE, defined as the duration of antinociception. Durationof antinociception, an index of total antinociception, was used toassess antinociception relative to respiratory depression for morphineand analogs.

Motor coordination was tested on a ROTOMEX-5 rotorod apparatus (ColumbusInstruments, Columbus, Ohio). Cumulative doses were given as describedin the antinociception section to produce >90% MPE on the TF test. Onlyrats remaining on the rotorod for 180 seconds during training weretested, allowing determination of % Maximum Possible Inhibition (% MPI)of motor coordination as [100−(latency to fall/180×100)].Antinociceptive ED₅₀ values were calculated by nonlinear regression. Anindex of motor impairment relative to antinociception was calculatedfrom the area under the curve (AUC) for MPI/AUC for MPE.

Tolerance was assessed by determining ED₅₀s before and after i.t. druginfusions for 7 days. Cumulative dosing with quarter-log increases every20 min were followed by a tail-flick test 15 min after injection.Immediately after the test in naïve rats, osmotic minipumps (ALZET model2001, Durect Corp, Cupertino, Calif.) filled with vehicle, morphine, oranalog and primed in 0.9% saline at 37° C. for 16 h, were implanted s.c.and connected to a PE-8 (0.008 inch I.D.) i.t. catheter. The pumpsdelivered 8× the ED₅₀/hr (2 μg/hr for morphine, 0.056 μg-0.075 μg/hr foranalog) for 7 days (53). A second dose-response curve was generated onday 7. ED₅₀ values are presented along with the shift in ED₅₀ thatprovides an index of relative tolerance.

Hyperalgesia relative to morphine was determined at baseline and on day7 with separate TF scores in which the heat intensity was set to evoke abaseline response >10 sec (cutoff 20 sec) to detect decreased latencies(increased sensitivity).

Immunohistochemistry: Animals from the tolerance experiment were deeplyanesthetized with ketamine/xylazine (85/10 mg/kg) and perfusedtranscardially with 0.1M PBS followed by 4% paraformaldehyde. Spinalcords were post-fixed overnight at 4° C., cryoprotected in 30%sucrose/0.1 M PBS for 48 hr, and sectioned on a freezing microtome at 40μm. After 2 washes in PBS and blocking with 5% normal goat serum/0.3%Triton X-100, sections were incubated in primary antibody; GFAP (1:1000,ab7779, Abcam, Cambridge, Mass.), Iba1 (1:1000, #019-19741, Wako,Richmond, Va.), pp38 (1:100, #4511, Cell Signaling Technology, Danvers,Mass.), OX-42 (1:100, #CBL1512, Millipore, Temecula, Calif.), or CGRP(1:1000, T-4032, Peninsula Labs, San Carlos, Calif.) for 24 h at 4° C.on a slow rocker. The tissue was then washed twice, re-blocked,incubated in donkey anti-rabbit secondary antibody conjugated to ALEXA488 dye (1:500 for GFAP, Iba1, CGRP, and 1:200 for pp38, #A21206Invitrogen, Eugene, Oreg.) or ALEXA 594 dye (1:500, #A21203, Invitrogen)for 2 hours (hrs) at room temperature (RT), washed, and slide-mountedwith PROLONG GOLD antifade reagent (Life Technologies, Grand Island,N.Y.). GFAP- and Ibal-immunoreactivities in lamina I-V of dorsal hornsegments L4-L5 were quantified on a NIKON microscope with a HAMMAMATSUcamera and NIH IMAGEJ software. Images containing lamina I-II of thespinal dorsal horn were analyzed for CGRP and OX-42 integrated densityusing IMAGEJ software (50). A blinded observer determined integrateddensity by thresholding the images using the default IMAGEJ algorithm toreduce background and include positively stained cells. Integrateddensity in the Region of Interest (ROI) is equal to the product of areaand mean gray value. The mean gray value represents the sum of theintensity values/number of pixels for all pixels above the threshold inthe ROI. This method controls for differences in background betweenslices and subjects. For quantification of pp38, an observer blinded totreatment manually counted punctate immunoreactive cells. Forco-labeling experiments, primary antibody against P2X7 receptors (P2X7R;1:100, #APR-008, Alamone Labs, Jerusalem, Israel) was incubated withOX-42 overnight. Tissue was washed and re-blocked as described above,and finally incubated with appropriate secondary antibodies ALEXA 488dye (1:500) and ALEXA 594 dye (1:500) before washing and mounting.Quantification of P2X7R and OX-42 co-labeling was performed using NIKONprojection images constructed from 1 μm thick image stacks from laminaI-II. The number of OX-42 positive cells and P2X7R/OX-42 co-labeledcells were counted to determine percent co-labeling (27). A total of 5-6rats per group and 4-6 slices/rat were quantified for all experiments.Representative confocal images were generated on a LEICA SP2 AOBSmicroscope.

Conditioned Place Preference (CPP):

Animals were allowed to freely explore an apparatus with a smaller“start box” and two larger distinct compartments (gray vs. black andwhite stripes of equal luminance; TSE Systems, Chesterfield, Mo.). Meantime in each compartment for 2 morning and 2 afternoon sessions of 20min each was determined as baseline. For the next three days, theanimals were given an i.v. injection of drug and immediately confined toone compartment for 30 min, and vehicle was given in the other. The timeat which the drug or vehicle was given (a.m. or p.m.) wascounterbalanced, as was the compartment in which the drug was given(preferred or non-preferred, as determined during the baseline test).The unbiased apparatus and design allows for detection of both drugpreference and aversion. After three days of conditioning, the animalswere allowed free access to all compartments in the drug-free state for20 min in the morning and afternoon. The mean change in time spent inthe drug-paired compartment was determined, and a significant increaseon the post-conditioning test relative to that on the pre-conditioningtest was interpreted as a CPP, reflective of rewarding properties andpotential abuse liability. Equi-antinociceptive asymptotic doses ofmorphine and analogs producing >95% MPE were used in the CPP test. Dosesof morphine and Analog 4 were also tested at 0.25 log lower (producing60-80% MPE), and 0.25 log higher than the maximal antinociceptive dose.

Self-administration (SA) tests were conducted in SA chambers (MEDAssociates, St. Albans, Vt.) containing an inactive lever and an activelever that regulated a computer-controlled infusion pump outside eachchamber. Infusions delivered through TYGON tubing in a balanced armswivel and metal spring leash allowed the animal free movement andprotected the infusion line. The protocol involved 7 sessions of 12-houraccess to saline or drug during the dark cycle (55). At the start ofeach 12-hour session, all rats received one priming injection equivalentto the same dose available during the session. The initial requirementof 1 active lever press per infusion (fixed ratio 1, FR1) was escalatedon days 3, 5 and 7 to FR2, 3 and 5, respectively. When the FRrequirement was completed, an infusion occurred along with a 10 sec timeout period when the stimulus lamp turned off and no additional infusionswere possible. Pressings on the active lever that resulted in aninfusion or occurred during the 10-sec time out period were analyzed andcompared to inactive lever responding. Active lever pressings/12 h,number of infusions/12 h, and intake (mg/kg/12 h) were analyzed fromdata averaged from the FR3-5 sessions to compare SA at high FR workloadrequirements to obtain infusion. In 12-hour variable dose studies, SAsessions were conducted for 12 hours per day using a descending doseparadigm in which 0.75, 0.3, 0.1, and 0 mg/kg/infusion of morphine oranalogs were available on days 1-2, 3-4, 5-7, and 8-10, respectively.

Alleviation of chronic pain by the analogs was tested in the sparednerve injury (SNI) model. At the site of the trifurcation of the leftsciatic nerve, the common peroneal and tibial branches were tightlyligated and transected, leaving the sural branch intact. Painsensitivity was assessed by applying pressure to the lateral edge of thehindpaw with a Randall-Selitto device. A baseline measure was takenbefore surgery and at 7 to 10 days postsurgery. Drugs were thenadministered intrathecally in cumulative doses chosen to produce fullalleviation of the hyperalgesia. Duration of pain alleviation (>vehicle)was assessed at 20 min intervals.

Data analysis: Data were analyzed by analysis of variance (ANOVA)followed, when appropriate, by Bonferroni, Newman-Kuels, or Dunnettpost-tests using GRAPHPAD PRISM software (GraphPad, San Diego, Calif.).Cumulative antinociceptive data from the tolerance study was analyzed bynon-linear regression to generate ED₅₀ values. Drug tolerance ED₅₀values were determined after acute and chronic administration.Immunohistochemical analysis of cell counts, integrated density, orco-labeling was conducted by blinded observers. Drugs were coded duringin vivo experiments and tested by blinded observers. All data ispresented as the mean±S.E. with 95% confidence limits. Differences wereconsidered statistically significant when p<0.05.

Results

EM Analogs Bind Selectively with High Affinity and Efficacy at Mu OpioidReceptors.

Analog structures are shown in FIG. 11, Panel A. Binding assays withcloned human opioid receptors showed that all four analogs hadselectivity and subnanomolar affinity for MOR (FIG. 11, Panel B). Analog4 showed the highest selectivity. In ³⁵5-GTPγS assays the analogs showedfull agonism at MOR, had greater potency than morphine, DAMGO and EMs,and remarkable selectivity for MOR activation (>100000 and >3000-foldhigher concentrations were required for delta or kappa activation).Interestingly, Analogs 1, 3 and 4 showed high efficacy delta antagonismat sub-micromolar concentrations. In vivo selectivity was alsodemonstrated: FIG. 11, Panel C shows that antinociceptive effects of allfour analogs were significantly blocked by naloxone (1 mg/kg), but notby antagonists for DOR (NTI) or KOR (nor-BNI) at doses (1 mg/kg) knownto antagonize selective agonists for those receptors after i.v.injection in the rat (26,30).

EM Analogs Show High Solubility and Stability.

The EM analogs showed favorable solubility (40, 20, 20 and 50 mg/mL inwater, 15, 15, 12, and 20 mg/mL in saline, and 90, 70, 50, and 50 mg/mLin 20% PEG400/saline for Analogs 1-4, respectively), and stable plasmahalf-life in vitro. While the parent endomorphins were metabolizedrapidly in rat plasma with a half-life of 5 min, Analogs 1-4 showedhalf-lives of 14, 46, 14, and 81 hr. The longer value for Analog 4relative to Analog 1 indicates that the glycine extension surprisinglyenhances stability. Analog 4 also was tested in human plasma and showeda half-life of about 150 hours (FIG. 12, Panel A). Stability in salineat 37° C. was >1 year (FIG. 12. Panel B), indicating a highly favorable“shelf life” and usefulness for long-term infusion.

Em Analogs Produce Potent, Long Lasting and Mu-Selective Antinociceptionafter Peripheral Administration.

The stability of the analogs translated to effectiveness afterperipheral administration as shown by potent and long lastingantinociception after intravenous (i.v.), subcutaneous (s.c.), and oraladministration. FIG. 12, Panels C-E illustrate these results for Analog4. At doses producing maximal % MPE, the durations of antinociceptionfor i.v. (FIG. 12, Panel C) s.c., (FIG. 12, Panel D) and oral (FIG. 12,Panel E) administration were 240, 80, and 100 min, respectively. Theduration of antinociception after i.v. injection in rat for morphine andall analogs is shown in FIG. 13, Panel C. FIG. 12, Panel D shows thatthe mu-selective antagonist β-FNA eliminated antinociception induced bys.c. administration of Analog 4 in mouse, consistent with theMOR-selective receptor binding data.

Respiratory Depression is Reduced or Absent after EM Analogs at DosesProducing Equal or Greater Duration of Antinociception Relative toMorphine.

Changes in MV over the 20 minute period of maximal drug effect areillustrated for morphine and Analog 4 in FIG. 13, Panel A, and averagechanges for all compounds in FIG. 13, Panel B. Duration ofantinociception determined from TF tests conducted at 20 min intervalsthereafter are shown in FIG. 13, Panel C. Morphine produced significantinhibition of MV at 5.6 and 10 mg/kg (FIG. 13, Panel A,B) along withabout 2-3 hr of antinociception (FIG. 13, Panel C). By contrast, afterEM analogs, respiratory inhibition was below, and duration ofantinociception (2-6 h) was at or above, that produced by morphine(dashed lines). The rank order for duration of antinociception wasAnalog 4>1>3>2>morphine. Analog 4 showed the clearest separation with norespiratory depression in the presence of significantly longerantinociception induced by lower doses. The results indicate that the EManalogs are clearly more effective and safer analgesics than morphine.

Motor Coordination is Impaired by Morphine but not EM Analogs.

Impairment of motor coordination relative to antinociception was testedin rotorod and TF tests. Analogs 2 and 4 produced significantly longerand greater total antinociception than morphine (FIG. 13, Panel D), weresignificantly more potent (FIG. 13, Panel F), and did not producesignificant motor impairment (FIG. 13, Panel D,E). Morphine showedsignificantly more impairment than vehicle and the analogs (FIG. 13,Panel D,E). Analog 4 provided the greatest duration of antinociceptionand the least motor impairment. Vehicle animals remained on the rotorodfor the 3 min maximum (no inhibition) and vehicle TF values (not shownfor clarity) were below 10% MPE at all time-points, indicating that thetesting sequence did not produce stress- or rotorod-inducedantinociception. Differences in motor impairment ratio (FIG. 13, PanelE) were significant with morphine (40.6+14.2) scores significantlygreater than those of vehicle (0.0), Analog 2 (8.0+8.0) and Analog 4(4.7+2.0), indicating a 5-fold and 8.6-fold better therapeutic ratio forAnalogs 2 and 4 relative to morphine.

Cognitive Function is Impaired by Morphine, but not Analog 4.

Tests of cognitive function (spatial memory) in the Morris Water Maze(MWM) revealed that Analog 4 shows a highly favorable profile relativeto morphine. After 4 days of training, an injection of morphine producedimpairment of spatial memory, as reflected by a significant increase inthe latency to, and average distance from, the platform. By contrast,Analog 4 did not produce significant impairment, even at doses thatprovided 2-3 times greater duration of antinociception than morphine.These results indicate an unexpected and superior therapeutic profile ofthe peptides of Formula I with regard to cognitive function relative tothe current standard opioid analgesic.

EM analogs access and activate CNS receptors. FIG. 13, Panel G showsthat i.c.v. low-dose (10 μg) injection of the blood-brain barrierimpermeable antagonist naloxone methiodide (Nlx-M) significantlyinhibited i.v. analog-induced antinociception (p<0.05). These resultsindicate that full antinociceptive effects require activation of CNSopioid receptors and that the relative lack of respiratory and motorimpairment by the analogs is not due to lack of CNS entry.

EM Analogs Produce Less Tolerance and Hyperalgesia than Morphine.

Tolerance and glial activation were tested in a well-characterized model(53,19). Cumulative intrathecal (i.t.) dosing of naïve rats showed thatEM analogs were initially 30-fold (60-fold on a molar basis) more potentthan morphine (FIG. 14, Panel A). After 7 days of infusion, the ED₅₀ ofmorphine shifted 38-fold. By contrast, EM analogs, tested at dosesmatched for antinociception, surprisingly produced a shift of only14-fold, demonstrating substantially less tolerance. Analog 4 wasselected for comparison to morphine for hyperalgesia. FIG. 15 shows that7-day treatment with morphine, but not EM Analog 4, inducedhyperalgesia.

Activation of Glial Cells by Morphine, but not EM Analogs.

Glial activation was assessed using markers for astrocytes (GFAP),microglia (Iba-1 and OX-42), and the activated microglial MAP kinasephospho-p38 (pp38) (FIG. 14, Panels B,C). Expression of all 3 markerswas significantly increased after morphine, but not after any of theanalogs. Expression of Iba-1 and pp38 after analogs was significantlybelow that of morphine.

Upregulation of CGRP by Morphine, but not EM Analogs.

CGRP induction has been implicated in glial activation and tolerance(56). FIG. 14, Panels D,E show CGRP staining was strongly upregulated inthe dorsal horn of the spinal cord by chronic infusion of morphine, butnot by any of the EM analogs.

Upregulation of P2X7 Receptors by Morphine, but not EM Analog 4.

Because upregulation of microglial P2X7R has been implicated intolerance (27,62) changes in this marker after morphine and Analog 4administration were tested. Morphine, but not Analog 4, upregulated P2X7receptors in microglial cells (FIG. 14, Panels F,G). Activated microgliacontained high levels of P2X7 receptor expression, confirmed byincreased microglial OX-42 that strongly co-labeled with P2X7 receptors.The high proportion of co-localization of upregulated P2X7 and activatedmicroglia after morphine (21% vs 5% after Analog 4 or vehicle) suggestsincreased ATP-P2X7-neurotrophin-cytokine mediated effects of morphine,but not Analog 4. In summary, EM analogs produced more potent acuteantinociception and, after chronic administration at equi-effectivedoses, induced substantially less tolerance and did not promote glialreactivity, CGRP upregulation or P2X7 induction compared to morphine.

Abuse liability was tested in two complementary paradigms, CPP and SA.CPP was compared using equally asymptotic antinociceptive doses thatproduced full antinociception (>95% MPE) 20 min after injection (FIG.16, Panel A), corresponding to the period of confinement to theconditioning side. These doses were determined as 3.2 mg/kg i.v. formorphine, 5.6, 3.2, 5.6, and 3.2 mg/kg for EM Analogs 1, 2, 3, and 4,respectively. FIG. 16, Panels B and C show that after 3 days ofconditioning, morphine, but not the analogs, produced a significantincrease in the time spent on the drug-paired side, a place preference.None of the compounds showed place aversion (decreased time on thedrug-paired side). Morphine and EM Analog 4 were also tested at 0.25 loghigher, and 0.25 log lower doses, but only morphine produced CPP. (FIG.16, Panel C).

While CPP has an advantage of testing for reward associations in thedrug-free state, SA more directly models drug consumption. Rats wereallowed to bar press for i.v. morphine, EM analogs or vehicle for 12h/day for 7 days with an escalating fixed ratio schedule. As the effortrequired to receive the drug increased from 1 to 5 presses per infusion(FIG. 16, Panel D), rats significantly escalated bar pressings formorphine, but not for the analogs (0.75 mg/kg/infusion). FIG. 16, PanelE compares active drug lever presses to an inactive lever duringhigh-effort sessions with FR requirements set at 3-5 (e.g. 3-5 pressesrequired for 1 infusion). Morphine produced significantly higher activelever pressings than vehicle or the inactive lever, while pressings forall analogs were significantly reduced by comparison. As doses weregradually lowered (0.75, 0.3, and 0.1 mg/kg/infusion) across sessions,rats self-administering morphine increased responding, while rats givenaccess to EM analogs did not escalate lever pressings (FIG. 16, PanelF). At comparably antinociceptive infusion doses (FIG. 16, Panels G-I)of morphine and EM Analog 4, the same pattern emerged; pressings formorphine (1 and 3 mg/kg/infusion) were significantly elevated over theinactive lever, whereas none of the doses of EM Analog 4 (1 and 3mg/kg/infusion) were self-administered (FIG. 16, Panels G-I). Thus, inCPP and SA models, the EM analogs showed significantly reduced rewardproperties relative to morphine, consistent with low abuse liability.

Support for the concept that Analog 4 could also be useful for treatingsubjects with a history of abuse was demonstrated in a drugdiscrimination paradigm. Rats were trained to discriminate morphineinjections from vehicle to receive food pellets. When challenged withAnalog 4, rats responded on the morphine lever, indicating they coulddiscriminate the EM analog as more similar to morphine than vehicle. Incombination with the lack of self-administrations by Analog 4, thesedata convey a compelling case for a compound that effectivelysubstitutes for morphine, but does not produce reward, indicating strongpotential for opioid addiction pharmacotherapy.

EM analogs provide potent and prolonged alleviation of chronic pain. Asshown in FIG. 17, Panel A, Analogs 1-3, and especially Analog 4, provideunexpectedly potent and long-lasting relief of neuropathic pain inducedby the spared nerve injury (SNI) model in the rat. Alleviation ofmechanical hypersensitivity (scores significantly greater than thoseafter vehicle) lasted just over 2 hours (about 135 min) after morphineand 4 hours or more (>235 min) after administration of the EM analogs.An increase, relative to morphine, in duration of relief was significantat 135 min for Analog 2, at 100 min for Analog 1 (135-155 min), and atleast about 140 min (95-235+ min) for Analog 4. Although potencydifferences among the analogs were not significant, the analogs were onaverage 80-fold more potent than morphine (p<0.0001). On a molar basis,this represents an average of 180-fold greater potency. The analogs,especially Analog 4, also produced more potent and longer duration ofrelief than morphine after other forms of chronic pain includingpost-incisional (post-operative) and inflammatory pain induced byComplete Freund's Adjuvant (CFA). The foregoing examples areillustrative, but not exhaustive, as to the types of acute or chronicpain for which the peptides of Formula I are effective.

As a result of the success of Analog 4 in the multiple tests describedhere, several additional hexapeptides of Formula I and a variant with2,6-dimethyltyrosine (DMT) in place of the Tyr residue at position 1were synthesized and tested for antinociception in the tail flick testfollowing subcutaneous injection to mice. FIG. 18 illustrates that bothTyr and DMT were effective in position 1, preferential amino acids inposition 6 were Gly (Analog 4) and Arg. Several additional amino acidsin position 6 induced antinociception to a lesser extent compared to Glyand Arg.

DISCUSSION

The EM analogs tested here provide potent antinociception (FIG. 12,Panels C-E, FIG. 13, Panel D, FIG. 14, Panel A, and FIG. 15, Panel A)with reduction or absence of six major side effects of currently usedopioids: abuse liability, respiratory depression, impairment of motorcoordination, tolerance, hyperalgesia, and glial activation. While allanalogs proved superior to morphine against the side effects tested,some differences indicate that they may provide new tools forunderstanding analgesia vs. side-effects and their separation forclinical application. First, Analog 2 (SEQ ID NO: 2) (an EM2 analog)showed low motor impairment relative to that produced by morphine andtwo analogs of EM1, i.e., Analog 1 (SEQ ID NO: 1) and Analog 3 (SEQ IDNO: 5). The latter analogs, however, showed preferable profiles inrespiratory and abuse liability tests. Analog 4 (SEQ ID NO: 4), based onthe EM1 structure, showed that remarkably low motor impairment, as wellas favorable respiratory and abuse liability profiles, could be achievedin a single molecule. Analog 4 proved unexpectedly superior in tests ofrespiratory depression, motor impairment (FIG. 13), and abuse liability(FIG. 14), while producing the most potent antinociceptive effects (FIG.13, Panels C and D). All four analogs produced far less tolerance thanmorphine (FIG. 14, Panel A) and did not induce glial cell (FIG. 14,Panel B) or CGRP (FIG. 14, Panel D) activation. While morphineupregulated microglial P2X7 receptors, Analog 4 did not (FIG. 14, PanelF). Compared to currently available opioids, the prolongedantinociception and lack of adverse effects from the analogs shown hereis unprecedented.

Analogs of Endomorphins.

The parent compounds of these analogs, the endomorphins (EMs) (23),showed early promise for a profile of potent analgesia with reduced sideeffects (29,22). The linear native peptides, however, are metabolicallylabile. While “constant renewal” methods such as viral vector-baseddelivery of these peptides have provided prolonged pain relief (47,59),structural modifications are required for more stable drug-likecompounds. Numerous analogs of EMs have been developed with the goal ofoptimizing drug-like properties and avoiding side-effects(37,40,41,42,52). The approach described herein has focused oncyclization combined with D-amino acid substitution in a cyclicpentapeptide or hexapeptide side chain-to-side chain structure. Thisstrategy permits amidation or extension of the C-terminus, and providesgreater solubility and improved pharmacological profiles relative tolinear peptide or side chain to C-terminus cyclic structures that weredescribed previously. The resulting compounds of Formula I show highsolubility, stability and favorable bioavailability as suggested byactivity after peripheral (including oral) administration (FIG. 12) andpenetration of the blood-brain barrier, as shown by central antagonismof antinociception produced by peripheral administration of the analogs(FIG. 13, Panel G). These advantageous drug characteristics areaccompanied by the favorable spectrum of antinociception/side effectprofiles described above.

Opioid-induced respiratory depression can be deadly, but the analogsdescribed herein (especially Analogs 1 and 4) did not impair respirationeven at equi- or greater-antinociceptive doses compared to morphine.Analog 4 showed no significant respiratory depression at doses producingsignificantly longer antinociception than morphine. Analogs 2 and 4 alsodid not produce impairment of motor coordination, while rats givenmorphine were significantly impaired at equi-antinociceptive doses.Older adults who take opioids for pain have an increased risk for fallsand accidents. Opioids such as those shown here, that alleviate painwithout producing motor deficits, would be particularly useful for thispopulation.

The analogs described herein produced significantly less tolerance thanmorphine even though the antinociceptive effects were matched. Numerousmechanisms have been postulated for opioid tolerance. Some mechanisms ofrelevance to the current study include effects of agonist efficacy, muagonist/delta antagonist properties, and glial activation. Stevens etal. (53) showed that sufentanyl produced less tolerance than morphine atcomparably effective doses. They suggested that high efficacy agonistshave a lower receptor reserve requirement for maintainingantinociception, and therefore show less tolerance. The analogsdescribed herein are consistent with this hypothesis, since they arehigh efficacy agonists and show reduced tolerance. Combining a delta(DOR) antagonist with a MOR agonist reduces tolerance (1). This profilefor Analogs 1, 3 and 4 (FIG. 11, Panel B) could contribute to therelative lack of tolerance. However, since the delta antagonist effectof the analogs is of high efficacy but low potency, further studieswould be required to establish the relative potency ratio of the MORagonism/DOR antagonism required to affect tolerance. In addition, Analog2 showed reduced tolerance despite a lack of DOR antagonism, indicatingthe likely involvement of multiple mechanisms and the importance of invivo tests for assessing this complex process.

A potentially important contributing mechanism to the reduced toleranceproduced by the analogs described herein is differential glialactivation. Many studies show that morphine activates glia through avariety of mechanisms, resulting in production of proinflammatorycytokines [IL-1, IL-6, IL-18, TNFα (27,39,49)] and PG, ATP, NO, and ROS(57), which are also “pronociceptive” and contribute to morphinetolerance and hyperalgesia (28,39,49,51,19,57,58). Phosphorylation ofmicroglial p38 (pp38), which induces many of these pronociceptivemolecules (38), plays a key role in tolerance paradigms (28,34,38,56).

While morphine-induced glial activation is well-established, themechanism is the subject of considerable debate. Three leadinghypotheses are (1) activation of a non-opioid site on microglialTLR4/1VID-2 receptors (38,58), (2) direct action at MOR on microgliathat stimulates purinergic (P2X) receptor signaling (31,35,62), and (3)the release of proinflammatory CGRP that activates glial-neuroninflammatory crosstalk (56). All three of these proposed mechanismsultimately converge onto the p38 pathway to induce the release ofpronociceptive molecules, so a crucial finding here is that chronicexposure to the analogs did not change pp38 levels while morphinesignificantly upregulated pp38. In addition, EM Analog 4 unexpectedlydid not change CGRP levels or P2X7 receptor (P2X7R) expression, incontrast to morphine which robustly increased the “activated” microgliaphenotype that strongly co-labeled with P2X7Rs. This is consistent withstudies showing direct CGRP (56) and P2X7R (27,62) involvement in thedevelopment of tolerance to morphine. Hence, the reduced tolerance andhyperalgesia after EM analogs may be partly due to less glialreactivity. The present study is the first to our knowledge in whichMOR-selective agonists did not induce glial, pp38, CGRP or P2X7Ractivation, while morphine, at the same antinociceptive effectiveness,produced significant activation.

Morphine-induced glial reactivity raises a host of issues concerningexacerbation of conditions associated with inflammation, includingpost-operative-induced (34), nerve injury-induced (34,43,49) andinflammation-induced (43) pain, traumatic brain injury (45),neurodegenerative diseases (32), and advancing age (25). Pain therapy inthese conditions would be best served by opioids shown here that do notexacerbate inflammatory responses. The discovery of opioid inducedproinflammatory responses has led to efforts to developanti-inflammatory medications as adjuncts to opioid treatment to enhanceanalgesia and reduce tolerance. The compounds of Formula I provide aclearly preferable approach, in that immune modulators would not berequired.

In addition to tolerance, differential glial activation may contributeto the differences between the analogs and morphine in other sideeffects. The microglial cell inhibitor minocycline blocked therespiratory depressive effects of morphine at similar doses and timeframe used here (36), but see (63). Several studies have linked glialreactivity to morphine-induced reward behaviors. Morphine-induced CPPwas associated with increased expression of Iba1 and pp38 in the nucleusaccumbens (NAc) (61). Systemic (36) and intra-accumbens (61) minocyclineblocked morphine-induced CPP, and intra-NAc injection of media fromcultured astrocytes potentiated CPP for morphine (46).

Although numerous EM analogs have been developed, abuse liabilityeffects have not been reported previously. Because of the importance ofthis side-effect, the analog compounds described herein were tested inboth conditioned place preference (CPP) and self-administration (SA)paradigms. Tested at a range of physiologically relevant doses, Analog 4did not produce CPP or aversive behaviors. Morphine promoted rewardeffects under long-term access SA conditions, the most sensitive SAparadigm (55), while the analogs did not. A recent review of ratself-administration studies showed that they were concordant with atleast one of two human clinical indicators of abuse liability in 64 of71 (90.1%) drug cases (48). Based on this correlation, the likelihood ofabuse in humans is low for the analogs. Circumventing abuse liability isa major advance, since most available opioids produce rewarding effectsin CPP and/or SA models (48,54).

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

REFERENCES

The following references are referred to in this application and areincorporated herein by reference in their entirety:

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What is claimed is:
 1. A cyclic peptide of Formula I: (I)H-Tyr-cyclo[X₁-X₂-X₃-X₄]-X₅,

wherein X₁ and X₄ each independently is an acidic D-amino acid or abasic D-amino acid; X₂ and X₃ each independently is an aromatic aminoacid; X₅ is selected from the group consisting of Ala-NHR, Arg-NHR,Asn-NHR, Asp-NHR, Cys-NHR, Glu-NHR, Gln-NHR, Gly-NHR, His-NHR, Ile-NHR,Leu-NHR, Met-NHR, Orn-NHR, Phe-NHR, Pro-NHR, Ser-NHR, Thr-NHR, Trp-NHR,Tyr-NHR, and Val-NHR, wherein R is H or an alkyl group; and there is anamide bond between an amino group and a carboxylic acid group on sidechains of amino acids X₁ and X₄; with the proviso that when X₁ is anacidic amino acid, then X₄ is a basic amino acid; and when X₁ is a basicamino acid, then X₄ is an acidic amino acid.
 2. The peptide of claim 1wherein: (i) X₁ is selected from the group consisting of D-Lys andD-Orn; and X₄ is selected from the group consisting of D-Asp, D-Glu,Asp, and Glu; or (ii) X₁ is selected from the group consisting of D-Aspand D-Glu; and X₄ is selected from the group consisting of D-Lys, D-Orn,Lys, and Orn.
 3. The peptide of claim 1, wherein: X₂ is selected fromthe group consisting of Trp, Phe, and N-alkyl-Phe, wherein the alkylgroup of N-alkyl-Phe comprises 1 to about 6 carbon atoms; and X₃ isselected from the group consisting of Phe, D-Phe, and p-Y-Phe, wherein Yis NO₂, F, Cl, or Br.
 4. The peptide of claim 3, wherein X₂ isN-methyl-Phe.
 5. The peptide of claim 3, wherein X₃ is p-Cl-Phe.
 6. Thepeptide of claim 1, wherein R is H.
 7. The peptide of claim 1, wherein Ris H and X₅ is Gly-NH₂ or Val-NH₂.
 8. The peptide of claim 1, whereinthe alkyl group is a methyl, ethyl, propyl, isopropyl, butyl, isobutyl,pentyl, isopentyl, hexyl, isohexyl, heptyl, or isoheptyl group.
 9. Thepeptide of claim 1, selected from the group consisting of:(SEQ ID NO: 3) Tyr-cyclo[D-Lys-Trp-Phe-Glu]-Gly-NH₂, (SEQ ID NO: 4)Tyr-cyclo[D-Glu-Phe-Phe-Lys]-Gly-NH₂ and (SEQ ID NO: 7)Tyr-cyclo[D-Orn-Phe-p-Cl-Phe-Asp]-Val-NH₂.


10. A cyclic peptide of Formula I: (I) H-Tyr-cyclo[X₁-X₂-X₃-X₄]-X₅,

wherein (i) X₁ is selected from the group consisting of D-Lys and D-Orn;and X₄ is selected from the group consisting of D-Asp, D-Glu, Asp, andGlu; or (ii) X₁ is selected from the group consisting of D-Asp andD-Glu; and X₄ is selected from the group consisting of D-Lys, D-Orn,Lys, and Orn; X₂ is selected from the group consisting of Trp, Phe, andN-alkyl-Phe, wherein the alkyl group of N-alkyl-Phe comprises 1 to about6 carbon atoms X₃ is selected from the group consisting of Phe, D-Phe,and p-Y-Phe, wherein Y is selected from the group consisting of NO₂, F,Cl, and Br X₅ is selected from the group consisting of Ala-NH₂, Arg-NH₂,Asn-NH₂, Asp-NH₂, Cys-NH₂, Glu-NH₂, Gln-NH₂, Gly-NH₂, His-NH₂, Ile-NH₂,Leu-NH₂, Met-NH₂, Orn-NH₂, Phe-NH₂, Pro-NH₂, Ser-NH₂, Thr-NH₂, Trp-NH₂,Tyr-NH₂, and Val-NH₂; and there is an amide bond between an amino groupand a carboxylic acid group on side chains of amino acids X₁ and X₄. 11.The cyclic peptide of claim 10, wherein X₅ is Gly-NH₂.
 12. The cyclicpeptide of claim 10, wherein X₁ is selected from the group consisting ofD-Lys and D-Orn; X₄ is selected from the group consisting of D-Asp andD-Glu; and X₅ is Gly-NH₂.
 13. The cyclic peptide of claim 10, whereinthe peptide has the amino acid sequence of SEQ ID NO:
 4. 14. A cyclicpeptide of Formula I: (I) H-Tyr-cyclo[X₁-X₂-X₃-X₄]-X₅,

wherein X₁ is an acidic or basic D-amino acid, and X₄ is a basic oracidic amino acid; X₂ is Trp; X₃ is selected from the group consistingof Phe, D-Phe, and p-Y-Phe, wherein Y is selected from the groupconsisting of NO₂, F, Cl, and Br; X₅ is Gly-NH₂; and there is an amidebond between an amino group and a carboxylic acid group on side chainsof amino acids X₁ and X₄; with the proviso that when X₁ is an acidicamino acid, then X₄ is a basic amino acid; and when X₁ is a basic aminoacid, then X₄ is an acidic amino acid.
 15. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and the peptide ofclaim
 1. 16. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and the peptide of claim
 10. 17. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and thepeptide of claim
 14. 18. A method of treating pain comprisingadministering to a subject an analgesic amount of the peptide ofclaim
 1. 19. A method of treating pain comprising administering to asubject an analgesic amount of the peptide of claim
 10. 20. A method oftreating pain comprising administering to a subject an analgesic amountof the peptide of claim
 14. 21. A method for treating a drug dependencecomprising administering to a subject a therapeutically effective amountof the peptide of claim
 1. 22. A method for treating a drug dependencecomprising administering to a subject a therapeutically effective amountof the peptide of claim
 10. 23. A method for treating a drug dependencecomprising administering to a subject a therapeutically effective amountof the peptide of claim
 14. 24. A method for treating a patient with ahistory of substance abuse comprising administering to a subject atherapeutically effective amount of the peptide of claim
 1. 25. A methodfor treating a patient with a history of substance abuse comprisingadministering to a subject a therapeutically effective amount of thepeptide of claim
 10. 26. A method for treating a patient with a historyof substance abuse comprising administering to a subject atherapeutically effective amount of the peptide of claim 14.